Oligomer-Protease Inhibitor Conjugates

ABSTRACT

The invention provides protease inhibitors that are chemically modified by covalent attachment of a water-soluble oligomer. A conjugate of the invention, when administered by any of a number of administration routes, exhibits characteristics that are different from the protease inhibitors not attached to the water-soluble oligomer.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)of U.S. Provisional Application Ser. No. 60/906,330, filed, Mar. 12,2007, which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention provides chemically modified small molecule proteaseinhibitors that possess certain advantages over small molecule proteaseinhibitors lacking the chemical modification. The chemically modifiedsmall molecule protease inhibitors described herein relate to and/orhave application(s) in (among others) the fields of drug discovery,pharmacotherapy, physiology, organic chemistry and polymer chemistry.

BACKGROUND OF THE INVENTION

Since the first cases of acquired immunodeficiency syndrome (AIDS) werereported in 1981, infection with human immunodeficiency virus (HIV) hasgrown to pandemic proportions, resulting in an estimated 65 millioninfections and 25 million deaths. See Aug. 11, 2006, MMWR 55(31):841-844(Center for Disease Control and Prevention). Protease inhibitorsrepresent an important class of compounds used to treat individualsinfected with HIV, although these compounds can also treat individualssuffering from other viral infections (e.g., Hepatitis C).

With respect to HIV, protease inhibitors act to inhibit the viralproteases that are necessary for the proteolytic cleavage of the gag andgag/pol fusion polypeptides necessary for the generation of infectiveviral particles. Thus, by inhibiting this proteolytic cleavage, proteaseinhibitors diminish the ability of larger HIV-fusion polypeptideprecursors to from the mature form of protein necessary for effectiveviral replication. McQuade et al. (1990) Science 247(4941):454-456.

Protease inhibitor-based therapy is acknowledged as an initial treatmentfor patients presenting symptomatic HIV disease and in non-symptomaticpatients after the CD4 cell count is below 350/μL but before a level of200/4. Hammer et al. (2006) JAMA 296(7):827-843. In such cases, aprotease inhibitor-based regimen will include a protease inhibitor(typically boosted with ritonavir) along with a combination of twonucleoside (or nucleotide) reverse transcriptase inhibitors. Id.

Although protease inhibitors serve an important role in treatingpatients suffering from HIV, their use has been hampered by challengesassociated with (among other things) extremely poor aqueous solubilityand extensive metabolism. One approach suggested to address thesedrawbacks includes preparing prodrug forms of protease inhibitors, suchas acyl and carbamotoyl glucose-containing prodrugs (Rouquayrol et al.(2001) Carbohydr. Res. 336:161-180) and relatively large PEG-basedprodrugs (Gunaseelan et al. (2004) Bioconjugate Chem. 15:1322-1333).Although potentially addressing some of the disadvantages associatedwith protease inhibitors, prodrug approaches necessarily result in thereturn of the original molecule, often along with its associateddrawbacks. For example, it is not believed that a prodrug approach wouldadequately solve the problems associated the extensive metabolismtypically observed with protease inhibitors.

The present invention seeks to address this and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula I.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula II.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula III.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula IV.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula V.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula VI.

In one or more embodiments, a compound is provided, the compoundcomprising a residue of a protease inhibitor covalently attached, eitherdirectly or through one or more atoms, via a stable linkage to awater-soluble, non-peptidic oligomer, wherein the protease inhibitor isencompassed by Formula VII.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

wherein:

-   -    is a residue of a small molecule protease inhibitor;    -   (a) is an integer having a value of one to three, inclusive;    -   X, in each occurrence, is a stable linkage; and    -   POLY, in each occurrence, is a water-soluble, non-peptidic        oligomer.

In one or more embodiments of the invention, a composition is provided,the composition comprising: a compound comprising a residue of aprotease inhibitor covalently attached, either directly or through oneor more atoms, via a stable linkage to a water-soluble, non-peptidicoligomer; and, optionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a composition is provided,the composition comprising:

-   -   (i) a compound having the following structure:

-   -   wherein:

-   -   -    is a residue of a small molecule protease inhibitor;        -   (a) is an integer having a value of one to three, inclusive;        -   X, in each occurrence is a stable linkage; and        -   POLY, in each occurrence, is a water-soluble, non-peptidic            oligomer; and

    -   (ii) optionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a residue of a proteaseinhibitor covalently attached, either directly or through one or moreatoms, via a stable linkage to a water-soluble, non-peptidic oligomer

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound having the following structure:

wherein:

-   -    is a residue of a small molecule protease inhibitor;    -   (a) is an integer having a value of one to three, inclusive;    -   X, in each occurrence is a stable linkage; and    -   POLY, in each occurrence, is a water-soluble, non-peptidic        oligomer.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to a small molecule protease inhibitor.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound having the followingstructure:

-   -   wherein:

-   -   -    is a residue of a small molecule protease inhibitor;        -   (a) is an integer having a value of one to three, inclusive;        -   X, in each occurrence, is a stable linkage; and        -   POLY, in each occurrence, is a water-soluble, non-peptidic            oligomer; and

    -   (ii) optionally, a pharmaceutically acceptable excipient.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent when read in conjunction withthe following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of amino acidresidues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer typically comprises one or moremonomers serially attached to form a chain of monomers. The oligomer canbe formed from a single monomer type (i.e., is homo-oligomeric) or twoor three monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 2 to about 50monomers, preferably from about 2 to about 30 monomers. The architectureof an oligomer can vary. Specific oligomers for use in the inventioninclude those having a variety of geometries such as linear, branched,or forked, to be described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” (also called an oligoethylene glycol) is one in whichsubstantially all (and more preferably all) monomeric subunits areethylene oxide subunits. The oligomer may, however, contain distinct endcapping moieties or functional groups, e.g., for conjugation. Typically,PEG oligomers for use in the present invention will comprise one of thetwo following structures: “—(CH₂CH₂O)_(n)—” or“—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whether the terminal oxygen(s)has been displaced, e.g., during a synthetic transformation. For PEGoligomers, “n” varies from about 2 to 50, preferably from about 2 toabout 30, and the terminal groups and architecture of the overall PEGcan vary. When PEG further comprises a functional group, A, for linkingto, e.g., a small molecule drug, the functional group when covalentlyattached to a PEG oligomer does not result in formation of (i) anoxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N).

An “end capping group” is generally a non-reactive carbon-containinggroup attached to a terminal oxygen of a PEG oligomer. Exemplary endcapping groups comprise a C₁₋₅ alkyl group, such as methyl, ethyl andbenzyl), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like.For the purposes of the present invention, the preferred capping groupshave relatively low molecular weights such as methyl or ethyl. Theend-capping group can also comprise a detectable label. Such labelsinclude, without limitation, fluorescers, chemiluminescers, moietiesused in enzyme labeling, colorimetric labels (e.g., dyes), metal ions,and radioactive moieties.

“Branched”, in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymers representingdistinct “arms” that extend from a branch point.

“Forked” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under ordinary physiological conditions. The tendency of a bond tohydrolyze in water under ordinary physiological conditions will dependnot only on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Such bonds aregenerally recognizable by those of ordinary skill in the art.Appropriate hydrolytically unstable or weak linkages include but are notlimited to carboxylate ester, phosphate ester, anhydrides, acetals,ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes under ordinary physiologicalconditions.

A “stable” linkage or bond refers to a chemical moiety or bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under ordinary physiologicalconditions to any appreciable extent over an extended period of time.Examples of hydrolytically stable linkages include but are not limitedto the following: carbon-carbon bonds (e.g., in aliphatic chains),ethers, amides, urethanes, amines, and the like. Generally, a stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under ordinary physiological conditions. Hydrolysis ratesof representative chemical bonds can be found in most standard chemistrytextbooks.

In the context of describing the consistency of oligomers in a givencomposition, “substantially” or “essentially” means nearly totally orcompletely, for instance, 95% or greater, more preferably 97% orgreater, still more preferably 98% or greater, even more preferably 99%or greater, yet still more preferably 99.9% or greater, with 99.99% orgreater being most preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, singlemolecular weight and defined number of monomers, as determined bychromatography or mass spectrometry. Monodisperse oligomer compositionsare in one sense pure, that is, substantially comprising moleculeshaving a single and definable number of monomers rather than severaldifferent numbers of monomers (i.e., an oligomer composition havingthree or more different oligomer sizes). A monodisperse oligomercomposition possesses a MW/Mn value of 1.0005 or less, and morepreferably, a MW/Mn value of 1.0000. By extension, a compositioncomprised of monodisperse conjugates means that substantially alloligomers of all conjugates in the composition have a single anddefinable number (as a whole number) of monomers rather than adistribution and would possess a MW/Mn value of 1.0005, and morepreferably, a MW/Mn value of 1.0000 if the oligomer were not attached tothe residue of the small molecule protease inhibitor. A compositioncomprised of monodisperse conjugates can include, however, one or morenonconjugate substances such as solvents, reagents, excipients, and soforth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a distribution, and whose distributionof molecular weights, when plotted as a number fraction versus molecularweight, appears as two separate identifiable peaks. Preferably, for abimodal oligomer composition as described herein, each peak is generallysymmetric about its mean, although the size of the two peaks may differ.Ideally, the polydispersity index of each peak in the bimodaldistribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000. By extension, a composition comprised of bimodal conjugatesmeans that substantially all oligomers of all conjugates in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution and would possessa MW/Mn value of 1.01 or less, more preferably 1.001 or less and evenmore preferably 1.0005 or less, and most preferably a MW/Mn value of1.0000 if the oligomer were not attached to the residue of the smallmolecule protease inhibitor agonist. A composition comprised of bimodalconjugates can include, however, one or more nonconjugate substancessuch as solvents, reagents, excipients, and so forth.

A “small molecule protease inhibitor” is broadly used herein to refer toan organic, inorganic, or organometallic compound typically having amolecular weight of less than about 1000 and having some degree ofactivity as a retroviral protease inhibitor. Small molecule proteaseinhibitors encompass oligopeptides and other biomolecules having amolecular weight of less than about 1000.

A “biological membrane” is any membrane, typically made from specializedcells or tissues, that serves as a barrier to at least some foreignentities or otherwise undesirable materials. As used herein a“biological membrane” includes those membranes that are associated withphysiological protective barriers including, for example: theblood-brain barrier (BBB); the blood-cerebrospinal fluid barrier; theblood-placental barrier; the blood-milk barrier; the blood-testesbarrier; and mucosal barriers including the vaginal mucosa, urethralmucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa,and so forth. Unless the context clearly dictates otherwise, the term“biological membrane” does not include those membranes associated withthe middle gastro-intestinal tract (e.g., stomach and small intestines).

A “biological membrane crossing rate,” as used herein, provides ameasure of a compound's ability to cross a biological membrane (such asthe membrane associated with the blood-brain barrier). A variety ofmethods can be used to assess transport of a molecule across any givenbiological membrane. Methods to assess the biological membrane crossingrate associated with any given biological barrier (e.g., theblood-cerebrospinal fluid barrier, the blood-placental barrier, theblood-milk barrier, the intestinal barrier, and so forth), are known inthe art, described herein and/or in the relevant literature, and/or canbe determined by one of ordinary skill in the art.

A “reduced rate of metabolism” in reference to the present invention,refers to a measurable reduction in the rate of metabolism of awater-soluble oligomer-small molecule drug conjugate as compared to rateof metabolism of the small molecule drug not attached to thewater-soluble oligomer (i.e., the small molecule drug itself) or areference standard material. In the special case of “reduced first passrate of metabolism,” the same “reduced rate of metabolism” is requiredexcept that the small molecule drug (or reference standard material) andthe corresponding conjugate are administered orally. Orally administereddrugs are absorbed from the gastro-intestinal tract into the portalcirculation and must pass through the liver prior to reaching thesystemic circulation. Because the liver is the primary site of drugmetabolism or biotransformation, a substantial amount of drug can bemetabolized before it ever reaches the systemic circulation. The degreeof first pass metabolism, and thus, any reduction thereof, can bemeasured by a number of different approaches. For instance, animal bloodsamples can be collected at timed intervals and the plasma or serumanalyzed by liquid chromatography/mass spectrometry for metabolitelevels. Other techniques for measuring a “reduced rate of metabolism”associated with the first pass metabolism and other metabolic processesare known in the art, described herein and/or in the relevantliterature, and/or can be determined by one of ordinary skill in theart. Preferably, a conjugate of the invention can provide a reduced rateof metabolism reduction satisfying at least one of the following values:at least about 30%; at least about 40%; at least about 50%; at leastabout 60%; at least about 70%; at least about 80%; and at least about90%. A compound (such as a small molecule drug or conjugate thereof)that is “orally bioavailable” is one that preferably possesses abioavailability when administered orally of greater than 25%, andpreferably greater than 70%, where a compound's bioavailability is thefraction of administered drug that reaches the systemic circulation inunmetabolized form.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced. An “alkenyl”group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, carboxy, acyl, cyano. The substituted alkyl groups maybe substituted once, twice or three times with the same or withdifferent substituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that can be included in the compositions ofthe invention in order to provide for a composition that has anadvantage (e.g., more suited for administration to a patient) over acomposition lacking the component and that is recognized as not causingsignificant adverse toxicological effects to a patient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosenfrom halo (F, Cl, Br, I), hydroxy, hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

An “aromatic-containing moiety” is a collection of atoms containing atleast aryl and optionally one or more atoms. Suitablearomatic-containing moieties are described herein.

For simplicity, chemical moieties are defined and referred to throughoutprimarily as univalent chemical moieties (e.g., alkyl, aryl, etc.).Nevertheless, such terms are also used to convey correspondingmultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, while an “alkyl” moietygenerally refers to a monovalent radical (e.g., CH₃—CH₂—), in certaincircumstances a bivalent linking moiety can be “alkyl,” in which casethose skilled in the art will understand the alkyl to be a divalentradical (e.g., —CH₂—CH₂—), which is equivalent to the term “alkylene.”(Similarly, in circumstances in which a divalent moiety is required andis stated as being “aryl,” those skilled in the art will understand thatthe term “aryl” refers to the corresponding divalent moiety, arylene).All atoms are understood to have their normal number of valences forbond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 forS, depending on the oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide athreshold level of active agent and/or conjugate in the bloodstream orin the target tissue. The precise amount will depend upon numerousfactors, e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterobifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, typically, but not necessarily, in theform of a water-soluble oligomer-small molecule drug conjugate, andincludes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a residue of a protease inhibitorcovalently attached, either directly or through one or more atoms, via astable linkage to a water-soluble, non-peptidic oligomer.

The present invention also provides a compound having the followingstructure:

wherein:

-   -    is a residue of a small molecule protease inhibitor;    -   (a) is an integer having a value of one to three, inclusive;    -   X, in each occurrence, is a stable linkage; and    -   POLY, in each occurrence, is a water-soluble, non-peptidic        oligomer.        The compounds of the invention are conjugates of an oligomer and        a protease inhibitor.

It is believed that an advantage of the conjugates of the presentinvention is their ability to retain some degree of protease activitywhile also exhibiting a decrease in metabolism. Although not wishing tobe bound by theory, it is believed that the oligomer-containingconjugates described herein—in contrast to the unconjugated “original”protease inhibitor—are not metabolized as readily because the oligomerserves to reduce the overall affinity of the compound to substrates thatcan metabolize protease inhibitors.

As indicated above, the compounds of the invention include a residue ofa small molecule protease inhibitor. The small molecule proteaseinhibitor is any small molecule that can reduce the activity of aretroviral protease. Assays for determining whether a compound(regardless of whether the compound is in conjugated form or not) is aprotease inhibitor are described infra.

Known compounds that act as small molecule protease inhibitors includethose selected from the following classes: azahexane derivatives; aminoacid derivatives; non-peptidic derivatives; pyranone compounds;pentan-1-amine derivatives; hexan-2-ylcarbamate derivatives; sulfonamidederivatives; and tri-substituted phenyl derivatives. Other smallmolecule protease inhibitors not necessarily belonging to any of theforegoing classes can also be used.

With respect to azahexane derivatives that are small molecule proteaseinhibitors, preferred azahexane derivatives have the following formula:

wherein:

R^(I1) is lower alkoxycarbonyl;

R^(I2) is secondary or tertiary lower alkyl or lower alkylthio-loweralkyl;

R^(I3) is phenyl that is unsubstituted or substituted by one or morelower alkoxy radicals, or C4-8 cycloalkyl;

R^(I4) is phenyl or cyclohexyl, each substituted in the 4-position byunsaturated heterocyclyl that is bonded by way of a ring carbon atom,has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selectedfrom the group nitrogen, oxygen, sulfur, sulfinyl (—SO—), and sulfonyl(—SO₂—) and is unsubstituted or substituted by lower alkyl or byphenyl-lower alkyl;

R^(I5) is secondary or tertiary lower alkyl or lower alkylthio-loweralkyl; and

R^(I6) is lower alkoxycarbonyl, and salts thereof.

A particularly preferred azahexane derivative is a compound of thefollowing formula:

which is also known as atazanavir. Atazanavir and other azahexanederivatives, as well as methods for their synthesis, are described inU.S. Pat. No. 5,849,911.

With respect to amino acid derivatives that are small molecule proteaseinhibitors, preferred amino acid derivatives have the following formula:

wherein:

R^(II1) is benzyloxycarbonyl or 2-quinolylcarbonyl, and pharmaceuticallyacceptable acid addition salts thereof. A particularly preferred aminoacid derivative is a compound of Formula II wherein R^(II1) is2-quinolylcarbonyl, also known as saquinavir. Such amino acidderivatives, as well as methods for their synthesis, are described inU.S. Pat. No. 5,196,438.

With respect to non-peptidic derivatives that are small moleculeprotease inhibitors, preferred non-peptidic derivatives have thefollowing structure:

wherein:

R^(III1) and R^(III2) are independently selected from hydrogen, andsubstituted and unsubstituted alkyl and aryl, and R^(III1) and R^(III2)may form a ring with G;

R^(III3) is selected from mercapto and substituted and unsubstitutedalkoxyl, aryloxyl, thioether, amino, alkyl, cycloalkyl, saturated andpartially saturated heterocycle, and aryl;

R^(III4), R^(III5), R^(III6), R^(III7), and R^(III8) are independentlyselected from hydrogen, hydroxyl, mercapto, nitro, halo, —O-J, wherein Jis a substituted or unsubstituted hydrolyzable group, and substitutedand unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfinyl,sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturatedheterocycle and aryl, and further wherein any of R^(III4), R^(III5),R^(III6), R^(III7), and R^(III8) may be a member of a spiro ring and anytwo of R^(III4), R^(III5), R^(III6), R^(III7), and R^(III8) may togetherbe members of a ring;

Y and G are independently selected from oxygen, —NH, —N-alkyl, sulfur,selenium, and two hydrogen atoms,

D is a carbon or nitrogen;

E is a carbon or nitrogen;

R^(III9) is selected from hydrogen, halo, hydroxyl, mercapto, andsubstituted and unsubstituted alkoxyl, aryloxyl, thioether, amino,alkyl, and aryl, wherein R^(III9) may form part of a ring;

A is a carbocycle or heterocycle, which is optionally furthersubstituted, and

B is a carbocycle or heterocycle, which is optionally furthersubstituted, or

a pharmaceutically acceptable salt thereof.

A particularly preferred non-peptidic derivative that is a smallmolecule protease inhibitor is a compound of the following formula:

which is also known as nelfinavir. Nelfinavir and other non-peptidicderivatives, as well as methods for their synthesis, are described inU.S. Pat. No. 5,484,926 and WO 95/09843.

With respect to pyranone compounds that are small molecule proteaseinhibitors, preferred pyranone compounds have the following structure:

wherein:

R^(IV4) is H; R^(IV2) is C₃₋₅ alkyl, phenyl-(CH₂)₂—,heterocycyl-SO₂NH—(CH₂)₂—, cyclopropyl-(CH₂)₂—, F-phenyl-(CH₂)₂—,heterocycyl-SO₂NH-phenyl-, or F₃C—(CH₂)₂—; or R^(IV1) and R^(IV2) takentogether are a double bond;

R^(IV3) is R^(IV4)—(CH₂)_(n)—CH(R^(IV5))—, H₃C—[O(CH₂)₂]₂—CH₂—, C₃₋₅alkyl, phenyl-(CH₂)₂—, heterocycyl-SO₂NH—(CH₂)₂—,(HOCH₂)₃C—NH—C(O)—NH—(CH₂)₃—, (H₂C)(H₂N)CH—(CH₂)₂—C(O)—NH—(CH₂)₃—,piperazin-1-yl-C(O)—NH—(CH₂)₃—,HO₃S(CH₂)₂—N(CH₃)—C(O)—(CH₂)₆—C(O)—NH—(CH₂)₃—, cyclopropyl-(CH₂)₂—,F-phenyl-(CH₂)₂—, heterocycyl-SO₂NH-phenyl-, or F₃—(CH₂)₂—; n′ is 0, 1or 2; R^(IV4) is phenyl, heterocycyl, cyclopropyl, H₃C—[O(CH₂)₂]₂—,heterocycyl-SO₂NH—, Br—, N₃—, or HO₃S(CH₂)₂—N(CH₃)—C(O)—(CH₂)₆—C(O)—NH—;R^(IV5) is —CH₂—CH₃, or —CH₂-cyclopropyl;

R^(IV6) is cyclopropyl, CH₃—CH₂—, or t-butyl;

R^(IV7) is —NR^(IV8)SO₂-heterocycyl, NR^(IV8)SO₂-phenyl, optionallysubstituted with R^(IV9), or —CH₂—SO₂-phenyl, optionally substitutedwith R^(IV9), or —CH₂—SO₂-heterocycyl; R^(IV8) is H, or —CH₃; R^(IV9) is—CN, —F, —OH, or —NO₂; wherein heterocycyl is a 5-, 6- or 7-memberedsaturated or unsaturated ring containing from one to three heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur; andincluding any bicyclic group in which any of the above heterocyclicrings is fused to a benzene ring or another heterocycle, optionallysubstituted with —CH₃, —CN, —OH, —C(O)OC₂H₅, —CF₃, —NH₂, or —C(O)—NH₂;or a pharmaceutically acceptable salt thereof.

A particularly preferred pyranone compound that is a small moleculeprotease inhibitor is a compound of the following formula:

which is also known as tipranavir. Tipranavir and other non-peptidicderivatives, as well as methods for their synthesis, are described inU.S. Pat. Nos. 6,147,095, 6,231,887, and 5,484,926.

With respect to pentan-1-amine derivatives that are small moleculeprotease inhibitors, preferred pentan-1-amine derivatives have thefollowing structure:

wherein:

R^(V0) is —OH or —NH₂;

Z^(V), in each instance, is independently O, S, or NH;

R^(V1) and R^(V2) are independently hydrogen or optionally substitutedC₁₋₄ alkyl, aryl, heterocycle, carbocyclic, —NH—SO₂C₁₋₃ alkyl, —O-aryl,—S-aryl, —NH-aryl, —O—C(O)-aryl, —S—C(O)-aryl, and —NH—C(O)-aryl, orR^(V1) and R^(V2) are joined together the form a monocyclic or bicyclicring system;

R^(V3) is hydrogen, C₁₋₄ alkyl, benzyl (substituted or unsubstituted);

J¹ and J² are independently —OH, —NH₂, or optionally substituted C₁₋₆alkyl, aryl, heterocycle, and carbocyclic, and

B is absent or selected from the group consisting of —NH—CH(CH₃)₂—C(O)—,—NH—CH(CH₃)₂—C(S)—, —NH—CH(CH₃)₂—C(NH)—, —NH—CH(CH₃)(CH₂CH₃)—C(O)—,—NH—CH(CH₃)(CH₂CH₃)—C(S)—, —NH—CH(CH₃)(CH₂CH₃)—C(NH)—,—NH—CH(phenyl)-C(O)—, —NH—CH(phenyl)-C(S)—, and —NH—CH(phenyl)-C(NH)—,

and pharmaceutically acceptable salts thereof.

A particularly preferred pentan-1-amine derivative that is a smallmolecule protease inhibitor is a compound of the following formula:

which is also known as indinavir. Indinavir and other pentan-1-aminederivatives, as well as methods for their synthesis, are described inU.S. Pat. No. 5,413,999 and European Patent Application No. EP 541 168.

With respect to hexan-2-ylcarbamate derivatives that are small moleculeprotease inhibitors, preferred hexane derivatives have the followingstructure:

wherein:

R^(VII) is monosubstituted thiazolyl, monosubstituted oxazolyl,monosubstituted isoxazolyl or monosubstituted isothiazolyl wherein thesubstituent is selected from (i) lower alkyl, (ii) lower alkenyl, (iii)cycloalkyl, (iv) cycloalkylalkyl, (v) cycloalkenyl, (vi)cycloalkenylalkyl, (vii) heterocyclic wherein the heterocyclic isselected from aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl,piperazinyl, morpholinyl, thiomorpholinyl, thiazolyl, oxazolyl,isoxazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyridazinyl andpyrazinyl and wherein the heterocyclic is unsubstituted or substitutedwith a substituent selected from halo, lower alkyl, hydroxy, alkoxy andthioalkoxy, (viii) (heterocyclic)alkyl wherein heterocyclic is definedas above, (ix) alkoxyalkyl, (x) thioalkoxyalkyl, (xi) alkylamino, (xii)dialkylamino, (xiii) phenyl wherein the phenyl ring is unsubstituted orsubstituted with a substituent selected from halo, lower alkyl, hydroxy,alkoxy and thioalkoxy, (xiv) phenylalkyl wherein the phenyl ring isunsubstituted or substituted as defined above, (xv) dialkylaminoalkyl,(xvi) alkoxy and (xvii) thioalkoxy;

n″ is 1, 2 or 3;

R^(VI2) is hydrogen or lower alkyl;

R^(VI3) is lower alkyl;

R^(VI4) and R^(4a) are independently selected from phenyl, thiazolyl andoxazolyl wherein the phenyl, thiazolyl or oxazolyl ring is unsubstitutedor substituted with a substituent selected from (i) halo, (ii) loweralkyl, (iii) hydroxy, (iv) alkoxy and (v) thioalkoxy;

R^(VI6) is hydrogen or lower alkyl;

R^(VI7) is thiazolyl, oxazolyl, isoxazolyl or isothiazolyl wherein thethiazolyl, oxazolyl, isoxazolyl or isothiazolyl ring is unsubstituted orsubstituted with lower alkyl;

R^(VI0) is hydrogen and Y^(VI) is —OH or X^(VI) is —OH and Y^(VI) ishydrogen, with the proviso that X^(VI) is hydrogen and Y^(VI) is —OHwhen Z^(VI) is —N(R^(VI8))— and R^(VI7) is unsubstituted and with theproviso that X^(VI) is hydrogen and Y^(VI) is —OH when R^(VI3) is methyland R^(VI7) is unsubstituted; and

Z^(VI) is absent, —O—, —S—, —CH²— or —N(R^(VI8))— wherein R^(VI8) islower alkyl, cycloalkyl, —OH or —NHR^(8a) wherein R^(8a) is hydrogen,lower alkyl or an amine-protecting group;

and pharmaceutically acceptable salts, esters or prodrug thereof.

A particularly preferred hexan-2-ylcarbamate derivative that is a smallmolecule protease inhibitor is a compound of the following formula:

which is also known as ritonavir.

Another particularly preferred hexan-2-ylcarbamate derivative that is asmall molecule protease inhibitor is a compound of the followingformula:

which is also known as lopinavir. Ritonavir, lopinavir and otherhexan-2-ylcarbamate derivatives, as well as methods for their synthesis,are described in U.S. Pat. No. 5,541,206 and WO 94/14436.

With respect to sulfonamide derivatives that are small molecule proteaseinhibitors, preferred sulfonamide derivatives have the followingstructure:

wherein:

A^(VII) is selected from the group consisting of H, Het, —R^(VIII)—Het,—R^(VIII)—C₁₋₆ alkyl, which may be optionally substituted with one ormore groups selected from the group consisting of hydroxy, C₁₋₄ alkoxy,Het, —O-Het, —NR^(VII2)—C(O)—N(R^(VII2))(R^(VII2)) and—C(O)—N(R^(VII2))(R^(VII2)); and —R^(VIII)—C₂₋₆ alkenyl, which may beoptionally substituted with one or more groups selected from the groupconsisting of hydroxy, C₁₋₄ alkoxy, Het, —O-Het,—NR^(VII2)—C(O)N(R^(VII2))(R^(VII2)) and —C(O)—N(R^(VII2))(R^(VII2));

each R^(VIII) is independently selected from the group consisting of—C(O)—, —SO₂—, —C(O)C(O)—, —O—C(O)—, —SO₂, —S(O)₂—C(O)— and—NR^(VII2)C(O)— and —NR^(VII2)—C(O)—C(O)—;

each Het is independently selected from the group consisting of C₃₋₇cycloalkyl; C₅₋₇ cycloalkenyl; C₆₋₁₀ aryl; and 5-7 membered saturated orunsaturated heterocycle, containing one or more heteroatoms selectedfrom N, N(R^(VII2)), O, S and S(O)_(n)—, wherein said heterocycle mayoptionally be benzofused; and wherein any member of said Het may beoptionally substituted with one or more substituents selected from thegroup consisting of oxo, —OR^(VII2), —R^(VII2), —N(R^(VII2)),—R^(VII2)—OH, —CN, CO₂R^(VII2), —C(O)N(R^(VII2))(R^(VII2)),SO₂—N(R^(VII2))(R^(VII2)), —N(R^(VII2))—C(O)—R^(VII2), —C(O)—R^(vIi2),—S(O)_(n)—R^(VII2), —OCF₃, —S(O)_(n)—Ar; methylenedioxy,—N(R^(vII2))—OO₂(R^(VII2))halo, —CF₃, —NO₂, Ar and —O—Ar;

each R^(VII2) is independently selected from the group consisting of Hand C₁₋₃ alkyl optionally substituted with Ar;

B^(VII), when present, is —N(R^(VII2))—C(R^(vII3))(R^(VII3))—C(O)—;

x′ is 0 or 1;

each R^(vII3) is independently selected from the group consisting of H,Het, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkyl and C₅₋₆ cycloalkenyl,wherein any member of said R^(vII3), except H, may be optionallysubstituted with one or more substituents selected from the groupconsisting of —OR^(VII2), —C(O)—NH—R^(VII2),—S(O)_(n)—N(R^(VII2))(R^(VII2)), Het, —CN, —SR^(VII2), —CO₂R^(VII2),NR^(VII2)—C(O)—R^(VII2);

each n′″ is independently 1 or 2;

D and D′ are independently selected from the group consisting of Ar;C₁₋₄ alkyl, which may be optionally substituted with one or more groupsselected from C₃₋₆ cycloalkyl, —OR^(VII2), —R^(II3), —O—Ar and Ar; C₂₋₄alkenyl, which may be optionally substituted with one or more groupsselected from the group consisting of C₃₋₆ cycloalkyl, —OR^(VII2),—R^(VII3), —O—Ar and Ar; C₃₋₆ cycloalkyl, which may be optionallysubstituted with or fused with Ar; and C₅₋₆ cycloalkenyl, which may beoptionally substituted with or fused with Ar;

each Ar is independently selected from the group consisting of phenyl;3-6 membered carbocyclic ring and 5-6 membered heterocyclic ringcontaining one or more heteroatoms selected from O, N, S, S(O)_(n)— andN(R^(VII2)), wherein said carbocyclic or heterocyclic ring may besaturated or unsaturated and optionally substituted with one or moregroups selected from the group consisting of oxo, —OR^(VII2), —R^(VII2),—N(R^(VII2))(R^(VII2)), —N(R^(VII2))—C(O)R^(VII2), —R^(VII2)—OH, —CN,—CO₂R^(VII2), —C(O)—N(R^(VII2))(R^(VII2)), halo and —CF₃;

E is selected from the group consisting of Het; O-Het; Het-Het;—O—R^(VII3); —NR^(VII2)R^(VII3); C₁₋₆ alkyl, which may be optionallysubstituted with one or more groups selected from the group consistingof R^(VII4) and Het; C₂₋₆ alkenyl, which may be optionally substitutedwith one or more groups selected from the group consisting of R^(VII4)and Het; C₃₋₆ saturated carbocycle, which may optionally be substitutedwith one or more groups selected from the group consisting of R^(VII4)and Het; and C₅₋₆ unsaturated carbocycle, which may optionally besubstituted with one or more groups selected from the group consistingof R^(VII4) and Het; and

each R^(VII4) is independently selected from the group consisting of−OR^(VII2), —C(O)—NHR^(VII2), SO₂—NHR^(VII2), halo,—NR^(VII2)—C(O)—R^(VII3) and —CN, and

pharmaceutically acceptable salts, esters or prodrug thereof.

A particularly preferred sulfonamide derivative that is a small moleculeprotease inhibitor is a compound of the following formula:

which is also known as amprenavir. Another particularly preferredsulfonamide derivative that is a small molecule protease inhibitor is acompound of the following formula:

Amprenavir, U-140690 and other sulfonamide derivatives, as well asmethods for their synthesis, are described in U.S. Pat. Nos. 5,732,490and 5,585,397, WO 93/23368, and WO 95/30670.

A particularly preferred prodrug form of a sulfonamide derivative is thephosphonooxy-based prodrug of the following formula:

which is known as fosamprenavir and pharmaceutically acceptable saltsthereof. Fosamprenavir and other sulfonamide derivatives, as well asmethods for their synthesis, are described in U.S. Pat. Nos. 6,514,953and 6,436,989.

With respect to tri-substituted phenyl derivatives that are smallmolecule protease inhibitors, preferred tri-substituted phenylderivatives have the following structure:

wherein:

R^(VIII1) is benzyl;

R^(VIII2) is benzyl or lower alkyl;

R^(VIII3) is lower alkyl; and

R^(VIII5) is

and pharmaceutically acceptable salts thereof. These and other smallmolecule protease inhibitors, as well as methods for their synthesis,are described in WO 97/21685.

As previously indicated, the small molecule protease inhibitor may notnecessarily be categorized within one of the aforementioned classes.Such small molecule protease inhibitors, however, can still beconjugated to a water-soluble, non-peptidic oligomer as describedherein. Nonlimiting additional small molecule protease inhibitorsinclude the compounds:

andrelated compounds, disclosed in WO 93/07128.

Still other small molecule protease inhibitors include:

and other others described in European Patent Application No. EP 580402.

Still other small molecule protease inhibitors include:

and other others described in WO 95/06061.

Still other small molecule protease inhibitors include:

and others described in EP 560268.

In some embodiments, it is preferred that the small molecule proteaseinhibitor is selected from the group selected from the group consistingof amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir,saquinavir, nelfinavir, ritonavir, tipranovir and darunavir.

Each of these (and other) protease inhibitor can be covalently attached(either directly or through one or more atoms) to a water-soluble,non-peptidic oligomer.

Small molecule drugs useful in the invention generally have a molecularweight of less than 1000 Da. Exemplary molecular weights of smallmolecule drugs include molecular weights of: less than about 950; lessthan about 900; less than about 850; less than about 800; less thanabout 750; less than about 700; less than about 650; less than about600; less than about 550; less than about 500; less than about 450; lessthan about 400; less than about 350; and less than about 300.

The small molecule drug used in the invention, if chiral, may be in aracemic mixture, or an optically active form, for example, a singleoptically active enantiomer, or any combination or ratio of enantiomers(i.e., scalemic mixture). In addition, the small molecule drug maypossess one or more geometric isomers. With respect to geometricisomers, a composition can comprise a single geometric isomer or amixture of two or more geometric isomers. A small molecule drug for usein the present invention can be in its customary active form, or maypossess some degree of modification. For example, a small molecule drugmay have a targeting agent, tag, or transporter attached thereto, priorto or after covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The small molecule protease inhibitor for coupling to a water-soluble,non-peptidic oligomer possesses a free reactive group, such as ahydroxyl, amide, carboxyl, thio, amino group, or the like (i.e.,“handle”) suitable for covalent attachment to the oligomer. In addition,the small molecule protease inhibitor can be modified by introduction ofa reactive group, preferably by conversion of one of its existingfunctional groups to a reactive group suitable for formation of a stablecovalent linkage between the oligomer and the drug. Both approaches areillustrated in the Experimental section.

The water-soluble, non-peptidic oligomer typically comprises one or moremonomers serially attached to form a chain of monomers. The oligomer canbe formed from a single monomer type (i.e., is homo-oligomeric) or twoor three monomer types (i.e., is co-oligomeric). Preferably, eacholigomer is a co-oligomer of two monomers or, more preferably, is ahomo-oligomer.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, saccharide or mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in the conjugateformula

) can have any of a number of different geometries. For example, thewater-soluble, non-peptidic oligomer can be linear, branched, or forked.Most typically, the water-soluble, non-peptidic oligomer is linear or isbranched, for example, having one branch point. Although much of thediscussion herein is focused upon poly(ethylene oxide) as anillustrative oligomer, the discussion and structures presented hereincan be readily extended to encompass any of the water-soluble,non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500 Daltons; below about 1450 Daltons; below about 1400Daltons; below about 1350 Daltons; below about 1300 Daltons; below about1250 Daltons; below about 1200 Daltons; below about 1150 Daltons; belowabout 1100 Daltons; below about 1050 Daltons; below about 1000 Daltons;below about 950 Daltons; below about 900 Daltons; below about 850Daltons; below about 800 Daltons; below about 750 Daltons; below about700 Daltons; below about 650 Daltons; below about 600 Daltons; belowabout 550 Daltons; below about 500 Daltons; below about 450 Daltons;below about 400 Daltons; below about 350 Daltons; below about 300Daltons; below about 250 Daltons; below about 200 Daltons; below about150 Daltons; and below about 100 Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges (end pointsfor each range provided are inclusive): between about 1 and about 30;between about 1 and about 25; between about 1 and about 20; betweenabout 1 and about 15; between about 1 and about 12; between about 1 andabout 10. In certain instances, the number of monomers in series in theoligomer (and the corresponding conjugate) is one of 1, 2, 3, 4, 5, 6,7, or 8. In additional embodiments, the oligomer (and the correspondingconjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20monomers in series. In yet further embodiments, the oligomer (and thecorresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or30 monomers in series. Thus, for example, when the water-soluble,non-peptidic polymer includes CH₃—(OCH₂CH₂)_(n)—, “n” is an integer thatcan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and can fall withinone or more of the following ranges: between about 1 and about 25;between about 1 and about 20; between about 1 and about 15; betweenabout 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to the smallmolecule protease inhibitor (in contrast to the step-wise addition ofone or more monomers to effectively “grow” the oligomer onto the smallmolecule protease inhibitor), it is preferred that the compositioncontaining an activated form of the water-soluble, non-peptidic oligomerbe monodispersed. In those instances, however, where a bimodalcomposition is employed, the composition will possess a bimodaldistribution centering around any two of the above numbers of monomers.Ideally, the polydispersity index of each peak in the bimodaldistribution, Mw/Mn, is 1.01 or less, and even more preferably, is 1.001or less, and even more preferably is 1.0005 or less. Most preferably,each peak possesses a MW/Mn value of 1.0000. For instance, a bimodaloligomer may have any one of the following exemplary combinations ofmonomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and soforth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5,3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10,and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9,6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and soforth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication2005/0136031.

The linker or linkage (through which the water-soluble, non-peptidicpolymer is attached to the small molecule protease inhibitor) at leastincludes a covalent bond, and often includes one or more atoms such asan oxygen, two atoms, or a number of atoms. A linker is typically but isnot necessarily linear in nature. The linkage, “X”

(in

), is a stable linkage, and is preferably also enzymatically stable.Preferably, the linkage “X” is one having a chain length of less thanabout 12 atoms, and preferably less than about 10 atoms, and even morepreferably less than about 8 atoms and even more preferably less thanabout 5 atoms, whereby length is meant the number of atoms in a singlechain, not counting substituents. For instance, a urea linkage such asthis, R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to have a chainlength of 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkagedoes not comprise further spacer groups.

In some instances, the linker “X” comprises an ether, amide, urethane,amine, thioether, urea, or a carbon-carbon bond. Functional groups suchas those discussed below, and illustrated in the examples, are typicallyused for forming the linkages. The linkage may less preferably alsocomprise (or be adjacent to or flanked by) spacer groups. Spacers aremost useful in instances where the bioactivity of the conjugate issignificantly reduced due to the positioning of the oligomer relativelyclose to the residue of the small molecule drug, wherein a spacer canserve to increase the distance between oligomer and the residue of thesmall molecule drug.

More specifically, in selected embodiments, a spacer moiety, X, may beany of the following: “—” (i.e., a covalent bond, that may be stable ordegradable, between the residue of the small molecule protease inhibitorand the water-soluble, non-peptidic oligomer), —O—, —NH—, —S—, —C(O)—,C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂,—CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—,—CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a group of atoms is notconsidered a spacer moiety when it is immediately adjacent to anoligomer segment, and the group of atoms is the same as a monomer of theoligomer such that the group would represent a mere extension of theoligomer chain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is typically formed by reaction of a functional group ona terminus of the oligomer (or one or more monomers when it is desiredto “grow” the oligomer onto the protease inhibitor) with a correspondingfunctional group within the protease inhibitor. Illustrative reactionsare described briefly below. For example, an amino group on an oligomermay be reacted with a carboxylic acid or an activated carboxylic acidderivative on the small molecule, or vice versa, to produce an amidelinkage. Alternatively, reaction of an amine on an oligomer with anactivated carbonate (e.g. succinimidyl or benzotriazyl carbonate) on thedrug, or vice versa, forms a carbamate linkage. Reaction of an amine onan oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, formsa urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol(alkoxide) group on an oligomer with an alkyl halide, or halide groupwithin a drug, or vice versa, forms an ether linkage. In yet anothercoupling approach, a small molecule having an aldehyde function iscoupled to an oligomer amino group by reductive amination, resulting information of a secondary amine linkage between the oligomer and thesmall molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4. Inaddition, the carbon atom alpha to the —C(O)H moiety can optionally besubstituted with alkyl.

Typically, the terminus of the water-soluble, non-peptidic oligomer notbearing a functional group is capped to render it unreactive. When theoligomer does include a further functional group at a terminus otherthan that intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional grouptypically comprises an electrophilic or nucleophilic group for covalentattachment to a small molecule, depending upon the reactive groupcontained within or introduced into the small molecule. Examples ofnucleophilic groups that may be present in either the oligomer or thesmall molecule include hydroxyl, amine, hydrazine (—NHNH₂), hydrazide(—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine. Most small moleculedrugs for covalent attachment to an oligomer will possess a freehydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, is 2-thiazolidine thione, etc., aswell as hydrates or protected derivatives of any of the above moieties(e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal,ketal, thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g. hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the protease inhibitor may not have a functional groupsuited for conjugation. In this instance, it is possible to modify the“original” protease inhibitor so that it does have the desiredfunctional group. For example, if the protease inhibitor has an amidegroup, but an amine group is desired, it is possible to modify the amidegroup to an amine group by way of a Hofmann rearrangement, Curtiusrearrangement (once the amide is converted to an azide) or Lossenrearrangement (once amide is concerted to hydroxamide followed bytreatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule proteaseinhibitor bearing a carboxyl group wherein the carboxyl group-bearingsmall molecule protease inhibitor is coupled to an amino-terminatedoligomeric ethylene glycol, to provide a conjugate having an amide groupcovalently linking the small molecule protease inhibitor agonist to theoligomer. This can be performed, for example, by combining the carboxylgroup-bearing small molecule protease inhibitor with theamino-terminated oligomeric ethylene glycol in the presence of acoupling reagent, (such as dicyclohexylcarbodiimide or “DCC”) in ananhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small moleculeprotease inhibitor bearing a hydroxyl group wherein the hydroxylgroup-bearing small molecule protease inhibitor is coupled to anoligomeric ethylene glycol halide to result in an ether (—O—) linkedsmall molecule conjugate. This can be performed, for example, by usingsodium hydride to deprotonate the hydroxyl group followed by reactionwith a halide-terminated oligomeric ethylene glycol.

In another example, it is possible to prepare a conjugate of a smallmolecule protease inhibitor bearing a ketone group by first reducing theketone group to form the corresponding hydroxyl group. Thereafter, thesmall molecule protease inhibitor now bearing a hydroxyl group can becoupled as described herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule protease inhibitor bearing an amine group. In oneapproach, the amine group-bearing small molecule protease inhibitor andan aldehyde-bearing oligomer are dissolved in a suitable buffer afterwhich a suitable reducing agent (e.g., NaCNBH₃) is added. Followingreduction, the result is an amine linkage formed between the amine groupof the amine group-containing small molecule protease inhibitor and thecarbonyl carbon of the aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a small moleculeprotease inhibitor bearing an amine group, a carboxylic acid-bearingoligomer and the amine group-bearing small molecule protease inhibitorare combined, typically in the presence of a coupling reagent (e.g.,DCC). The result is an amide linkage formed between the amine group ofthe amine group-containing small molecule protease inhibitor and thecarbonyl of the carboxylic acid-bearing oligomer.

Exemplary conjugates of the small molecule protease inhibitors ofFormula I include those having the following structures:

wherein for each of Formula I-Ca, Formula I-Cb and Formula I-Cc: X is astable linkage; POLY is a water-soluble, non-peptidic oligomer; and eachof R^(I1), R^(I2), R^(I3), R^(I4), R^(I5) and R^(I6) is as defined withrespect to Formula I.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structures:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of small molecule protease inhibitors of Formula IIinclude those having the following structures:

wherein, in each instance in which it appears: X is a stable linkage;POLY is a water-soluble, non-peptidic oligomer; and R^(II1) isbenzyloxycarbonyl or 2-quinolylcarbonyl.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structures:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula III include those having the following structures:

wherein, in each instance in where it appears: X is a stable linkage;POLY is a water-soluble, non-peptidic oligomer; and each of R^(III1),R^(III2), R^(III3), R^(III4), R^(III5), R^(III6), R^(III7), R^(III8), Y,G, D, E, R^(III9), A and B is as defined with respect to Formula III.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structure:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula IV include those having the following structure:

wherein: X is a stable linkage; POLY is a water-soluble, non-peptidicoligomer; and R^(IV1), R^(IV2), R^(IV3), R^(IV6) and R^(IV7) is asdefined with respect to Formula IV.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structure:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula V include those having the following structure:

wherein, in each instance where it appears: X is a stable linkage; POLYis a water-soluble, non-peptidic oligomer; and each of Z^(V), R^(V1),R^(V2), R^(V3), J¹, J² and B is as defined with respect to Formula V.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structure:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula VI include those having the following structure:

wherein: X is a stable linkage; POLY is a water-soluble, non-peptidicoligomer; R^(VI0) is H; and each of R^(VI1), n″, R^(VI3), R^(VI4),R^(4a) an Z^(VI) is as defined with respect to Formula VI.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structures:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula VII include those having the following structure:

wherein: X is a stable linkage; POLY is a water-soluble, non-peptidicoligomer; and each of A^(VI1), B^(VI1), x′D, D′ and E^(VI1) is asdefined with respect to Formula VII.

Preferred conjugates of small molecule protease inhibitors include thosehaving the following structures:

wherein, in each instance where it appears, n is an integer from 2 to30.

Exemplary conjugates of the small molecule protease inhibitors ofFormula VIII include those having the following structures:

wherein: X is a stable linkage; POLY is a water-soluble, non-peptidicoligomer; and each of R^(VIII1), R^(VIII2) and R^(III3) is as definedwith respect to Formula VIII.

Still further exemplary conjugates include those having the followingstructures (wherein, with respect to each structure, X is a stablelinkage and POLY is a water-soluble, non-peptidic oligomer):

wherein, in each instance in which it appears, X is a stable linkage,POLY is a water-soluble, non-peptidic oligomer, and Val is a residue ofvaline.

One of ordinary skill in the art, using routine experimentation, candetermine a best suited molecular size and linkage for reducingmetabolism by first preparing a series of oligomers with differentweights and functional groups and then obtaining the necessary clearanceprofiles by administering the conjugates to a patient and takingperiodic blood and/or urine sampling and testing for the presence andamount of metabolites. Once a series of metabolism profiles have beenobtained for each tested conjugate, a suitable conjugate can beidentified.

To determine whether the small molecule protease inhibitor or theconjugate of a small molecule protease inhibitor and a water-solublenon-peptidic polymer has anti-HIV activity, it is possible to test suchcompounds. Anti-HIV activity can be tested as described in theExperimental. In addition, Anti-HIV activity can be tested in a humanT-cell line by, for example, the method disclosed in Kempf et al. (1991)Antimicrob. Agents Chemother. 35(11):2209-2214, HIV-1_(3B) stock(10^(4.7) 50% tissue culture infection doses per ml) can be diluted100-fold and incubated with MT-4 cells at 4×10⁵ cells per ml for onehour at 37° C. (multiplicity of infection, 0.001 50% tissue cultureinfective dose per cell). The resulting culture is then washed twice,resuspended to 10⁵ cells per ml of medium, seeded in a volume of 1%dimethyl sulfoxide solution of compound in a series of half-log-unitdilutions in medium in triplicate. The virus control culture can betreated in an identical manner, except that no compound is added to themedium. The cell control is incubated in the absence of compound orvirus. Optical density (OD) is then measured at day 5 by using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in acolorimetric assay. See Pauwels et al. (1988) J. Virol Methods20:309-321. Virus and control OD values are averaged over sixdeterminations. Percent inhibition of HIV cytopathic effect (CPE) iscalculated by the following formula: [(average OD−virus control OD/(cellcontrol OD−virus control OD)]×100. Cytotoxicity is determined by theincubation in duplicate with serial dilutions of compound in the absenceof virus. Percent cytotoxicity is determined according to the followingformula: (average OD/cell control OD)×100. The EC₅₀ represents theconcentration of compound that gave 50% inhibition of the cytopathiceffect. The CClC₅₀ is the concentration of compound which gives a 50%cytotoxic effect. It is noted that when conjugation of thewater-soluble, non-peptidic oligomer occurs at the hydroxyl grouplocated at 26 position of saquinavir, no anti-HIV activity is measured.See Table 1, Example 3. While not wishing to be bound by theory, itappears that the availability of this hydroxyl group is required foractivity (a “binding hydroxyl group”). As a consequence, it is preferredin some embodiments that the conjugate lacks attachment of thewater-soluble, non-peptidic oligomer at a binding hydroxyl group. A“binding hydroxyl group” for any given protease inhibitor can bedetermined by one of ordinary skill in the art by, for example,experimental testing and/or by comparing the structure of the proteaseinhibitor of interest with the structure of saquinavir and determiningwhich hydroxyl group in the protease inhibitor corresponds to the“binding hydroxyl group” at position 26 in saquinavir.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, and the like. Binders are used to impart cohesive qualities to atablet, and thus ensure that the tablet remains intact. Suitable bindermaterials include, but are not limited to, starch (including corn starchand pregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, waxes, and natural andsynthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, microcrystalline cellulose, ethylcellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricantsare used to facilitate tablet manufacture, promoting powder flow andpreventing particle capping (i.e., particle breakage) when pressure isrelieved. Useful lubricants are magnesium stearate, calcium stearate,and stearic acid. Disintegrants are used to facilitate disintegration ofthe tablet, and are generally starches, clays, celluloses, algins, gums,or crosslinked polymers. Fillers include, for example, materials such assilicon dioxide, titanium dioxide, alumina, talc, kaolin, powderedcellulose, and microcrystalline cellulose, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form(typically as a lyophilizate or precipitate, which can be in the form ofa powder or cake), as well as formulations prepared for injection, whichare typically liquid and requires the step of reconstituting the dryform of parenteral formulation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions, eachtypically being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of

administration are also contemplated, such as pulmonary, nasal, buccal,rectal, sublingual, transdermal, and parenteral. As used herein, theterm “parenteral” includes subcutaneous, intravenous, intra-arterial,intraperitoneal, intracardiac, intrathecal, and intramuscular injection,as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature and/or can be determined experimentally. Generally, atherapeutically effective amount is an amount within one or more of thefollowing ranges: from 0.001 mg/day to 10000 mg/day; from 0.01 mg/day to7500 mg/day; from 0.10 mg/day to 5000 mg/day; from 1 mg/day to 4000mg/day; and from 10 mg/day to 2000 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that a reduction in first pass metabolism may be achieved relative tothe parent drug. Such a result is advantageous for many orallyadministered drugs that are substantially metabolized by passage throughthe gut. In this way, clearance of the conjugate can be modulated byselecting the oligomer molecular size, linkage, and position of covalentattachment providing the desired clearance properties. One of ordinaryskill in the art can determine the ideal molecular size of the oligomerbased upon the teachings herein. Preferred reductions in first passmetabolism for a conjugate as compared to the correspondingnonconjugated small drug molecule include: at least about 10%, at leastabout 20%, at least about 30; at least about 40; at least about 50%; atleast about 60%, at least about 70%, at least about 80% and at leastabout 90%.

Thus, the invention provides a method for reducing the metabolism of anactive agent. The method comprises the steps of: providing monodisperseor bimodal conjugates, each conjugate comprised of a moiety derived froma small molecule drug covalently attached by a stable linkage to awater-soluble oligomer, wherein said conjugate exhibits a reduced rateof metabolism as compared to the rate of metabolism of the smallmolecule drug not attached to the water-soluble oligomer; andadministering the conjugate to a patient. Typically, administration iscarried out via one type of administration selected from the groupconsisting of oral administration, transdermal administration, buccaladministration, transmucosal administration, vaginal administration,rectal administration, parenteral administration, and pulmonaryadministration.

Although useful in reducing many types of metabolism (including bothPhase I and Phase II metabolism) can be reduced, the conjugates areparticularly useful when the small molecule drug is metabolized by ahepatic enzyme (e.g., one or more of the cytochrome P450 isoforms)and/or by one or more intestinal enzymes.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031. All oligo(ethylene glycol) methyl ethersemployed in the Examples below were monodisperse and chromatographicallypure, as determined by reverse phase chromatography.

Example 1 Synthesis of PEG-Saquinavir Conjugates Conjugation at theSaquinavir Hydroxyl Group at C₂₆ Position

A. Synthesis of 26-m-PEG-3-O-Saquinavir (n=3)

NaH (60% in mineral oil, 72 mg, 1.8 mmole) was added into a solution ofsaquinavir free base (170 mg, 0.30 mmole) in dimethylformamide (5 ml).The mixture was stirred at room temperature under N₂ for 15 minutes,followed by the addition of Br—(CH₂CH₂O)₃—CH₃ (273 mg, 1.2 mmole) indimethylformamide (1 ml). The resulting solution was then heated at 50°C. under N₂ in an oil bath for four hours. All solvents were thenremoved by using a rotary evaporator. Pure 26-m-PEG-3-O-Saquinavir wasobtained by reverse phase preparative HPLC separation (76 mg, 0.093mmole, 31% isolated yield).

Synthesis of 26-m-PEG-7-O-Saquinavir (n=7)

NaH (60% in mineral oil, 108 mg, 2.7 mmole) was added into a solution ofsaquinavir free base (300 mg, 0.45 mmole) in dimethylformamide (10 ml).The mixture was stirred at room temperature under N₂ for 15 minutes,followed by the addition of Br—(CH₂CH₂O)₇—CH₃ (724 mg, 1.8 mmole) indimethylformamide (1 ml). The resulting solution was then heated at 50°C. under N₂ in an oil bath for four hours. All solvents were thenremoved by using a rotary evaporator. Pure 26-m-PEG-7-O-Saquinavir wasobtained by reverse phase preparative HPLC separation (100 mg, 0.10mmole, 22% isolated yield).

Example 2 Synthesis of PEG-Saquinavir Conjugates Conjugation at theSaquinavir Amide Group at C₁₅ Position

Synthesis of 15-m-PEG-7-NHCO-Saquinavir and 15-di-m-PEG-7-NCO-Saquinavir(n=7)

Synthesis of 26-MEM-O-Saquinavir. To an anhydrous tetrahydrofuran (−30mL) solution of saquinavir free base (530 mg, 0.79 mmol) at −30° C.under N₂, butyl lithium (1.58 mmol, 0.63 mL, 2.5 M in hexane) was addedthrough a syringe. After stirred at −30° C. for 5 minutes, MEMCl (118mg, 0.95 mmol) in ˜1 mL of anhydrous tetrahydrofuran was added. Thereaction solution was slowly warmed up to room temperature and keptovernight (18 hours). HPLC showed that almost all free saquinavir wasgone and 26-MEM-β-Saquinavir was formed in ˜90% yield. After separationby a reverse phase preparative HPLC, pure 26-MEM-O-Saquinavir wasobtained as a colorless solid.

Synthesis of 26-MEM-O-15-m-PEG-7-NHCO-Saquinavir. To a dimethylformamide(˜8 mL) solution of 26-MEM-O-Saquinavir (50 mg, 0.066 mmole) was addedsodium hydride (21 mg, 0.53 mmole, 60% in mineral oil). After stirringat room temperature for 15 minutes, Br—(CH₂CH₂O)₇—CH₃ (159 mg, 0.40mmole) in ˜1 mL of dimethylformamide was added. The reaction mixture wasstirred at room temperature under nitrogen for two days. HPLC showedthat 26-MEM-O-15-m-PEG-7-NHCO-Saquinavir was formed in ˜50% yield. Thereaction was then stopped by the addition of 0.1N hydrochloric acidsolution (˜3 mL) to destroy excess sodium hydride. All the solvents wereremoved by a rotary evaporator at 50° C. to give a sticky solid. Theproduct was not purified and was used as such in the next syntheticstep.

Synthesis of 15-m-PEG-7-NHCO-Saquinavir. The reaction mixture of26-MEM-O-15-m-PEG-7-NHCO-Saquinavir was dissolved in ˜10 mL of 2Nhydrochloric acid methanol solution. The solution was stirred at roomtemperature for four days. HPLC showed that all MEM protection groupswere removed and 15-m-PEG-7-NHCO-Saquinavir was formed in ˜50% yield.After the reverse phase preparative HPLC separation, pure15-m-PEG-7—NHCO-Saquinavir was obtained (35 mg, 0.035 mmole, 53%isolated yield), LC-MS: Calc: 993.2. Found: 993.5.

Synthesis of 26-MEM-O-15-di-m-PEG-7-NCO-Saquinavir. To adimethylformamide (˜5 mL) solution of 26-MEM-O-Saquinavir (30 mg, 0.040mmole) was added sodium hydride (26 mg, 0.64 mmole, 60% in mineral oil).After stirring at room temperature for 15 minutes, Br—(CH₂CH₂O)₇—CH₃ (96mg, 0.24 mmole) in ˜1 mL of dimethylformamide was added. The reactionmixture was stirred at room temperature under nitrogen for two days.HPLC showed that 26-MEM-O-15-di-m-PEG-7-NCO-Saquinavir was formed in˜23% yield. The reaction was then stopped by the addition of 0.1Nhydrochloric acid solution (˜3 mL) to destroy excess sodium hydride. Allthe solvents were removed by a rotary evaporator at 50° C. to give asticky solid. The product was not purified and was used as such for thenext synthetic step.

Synthesis of 15-di-m-PEG-7-NCO-Saquinavir. The reaction mixture of26-MEM-O-di-15-m-PEG-7-NCO-Saquinavir was dissolved in ˜10 mL of 2Nhydrochloric acid methanol solution. The solution was stirred at roomtemperature overnight. HPLC showed that all MEM protection groups wereremoved and 15-di-m-PEG-7-NCO-Saquinavir was formed in ˜37% yield. Afterthe reverse phase preparative HPLC separation, pure15-di-m-PEG-7-NCO-Saquinavir was obtained (11 mg, 0.0084 mmole, 21%isolated yield), LC-MS: Calc: 1315.6. Found: 1315.6.

Synthesis of 15-m-PEG-3-NHCO-Saquinavir (n=3)

Synthesis of 26-MEM-O-15-m-PEG-3-NHCO-Saquinavir. To a dimethylformamide(˜20 mL) solution of 26-MEM-O-Saquinavir (88 mg, 0.12 mmole) was addedsodium hydride (37 mg, 0.92 mmole, 60% in mineral oil). After stirringat room temperature for 15 minutes, Br—(CH₂CH₂O)₃—CH₃ (158 mg, 0.70mmole) in ˜1 mL of dimethylformamide was added. The reaction mixture wasstirred at room temperature under nitrogen overnight (˜23 hours). HPLCshowed that 26-MEM-O-15-m-PEG-3-NHCO-Saquinavir was formed in ˜47%yield. The reaction was then stopped by the addition of 0.1Nhydrochloric acid solution (˜3 mL) to destroy excess sodium hydride. Allthe solvents were removed by a rotary evaporator at 50° C. to give asticky solid. The product was not purified and was used as such for thenext synthetic step.

Synthesis of 15-m-PEG-3-NHCO-Saquinavir. The crude26-MEM-O-15-m-PEG-3—NHCO-Saquinavir product was dissolved in ˜30 mL of2N hydrochloric acid methanol solution. The solution was stirred at roomtemperature for four hours. HPLC showed that all MEM protection groupswere removed and 15-m-PEG-3-NHCO-Saquinavir was formed in ˜41% yield.After the reverse phase preparative HPLC separation, pure15-m-PEG-3—NHCO-Saquinavir was obtained (20 mg, 0.024 mmole, 20%isolated yield), LC-MS: Calc: 817.0. Found: 817.5.

Synthesis of 15-m-PEG-5-NHCO-Saquinavir (n=5)

Synthesis of 26-MEM-O-15-m-PEG-5-NHCO-Saquinavir. To a dimethylformamide(˜30 mL) solution of 26-MEM-O-Saquinavir (140 mg, 0.18 mmole) was addedsodium hydride (59 mg, 1.48 mmole, 60% in mineral oil). After stirringat room temperature for 15 minutes, Br—(CH₂CH₂O)₅—CH₃ (349 mg, 1.11mmole) in ˜1 mL of dimethylformamide was added. The reaction mixture wasstirred at room temperature under nitrogen for two days. HPLC showedthat 26-MEM-O-15-m-PEG-5-NHCO-Saquinavir was formed in ˜52% yield. Thereaction was then stopped by the addition of 0.1N hydrochloric acidsolution (˜3 mL) to destroy excess sodium hydride. All the solvents wereremoved by a rotary evaporator at 50° C. to give a sticky solid. Theproduct was not purified and was used as such for the next syntheticstep.

Synthesis of 15-m-PEG-5-NHCO-Saquinavir. The reaction mixture of26-MEM-O-15-m-PEG-5-NHCO-Saquinavir was dissolved in ˜15 mL of 2Nhydrochloric acid methanol solution. The solution was stirred at roomtemperature for four hours. HPLC showed that all MEM protection groupswere removed and 15-m-PEG-5-NHCO-Saquinavir was formed in ˜50% yield.After the reverse phase preparative HPLC separation, pure15-m-PEG-5—NHCO-Saquinavir was obtained (32 mg, 0.035 mmole, 20%isolated yield), LC-MS: Calc: 905.1. Found: 905.5.

Example 3 Evaluation for Anti-HIV-1 Efficacy in CEM-SS Cells

Compounds were tested at a 1.0 μM high-test concentration in DMSO.CEM-SS cells were passaged in T-75 flasks prior to use in the antiviralassay. On the day preceding the assay, the cells were split 1:2 in orderto assure they were in exponentional growth phase at the time ofinfection. Total cell and viability quantification was performed using ahemacytometer and trypan blue exclusion. Cell viability was greater than95% for the cells to be utilized in the assay. The cells wereresuspended at 5×10⁴ cells/mL in tissue culture medium and added to thedrug-containing microtiter plates in a volume of 50 μL.

The virus used was the lymphocytropic virus strain HIV-1_(RF). Thisvirus was obtained from the NIH AIDS research and Reference ReagentProgram and was grown in CEM-SS cells for the production of stock viruspools. For each assay, a pre-titered aliquot of virus was removed fromthe freezer and allowed to thaw slowly to room temperature in abiological safety cabinet. The virus was resuspended and diluted intotissue culture medium such that the amount of virus added to each wellin a volume of 50 μL was the amount determined to give approximately 90%cell killing in six days post-infection. TCID₅₀ calculations by endpointtitration in CEM-SS cells indicated that the multiplicity of infectionin these assays was approximately 0.01.

Each plate contains cell control wells (cells only), virus control wells(cells plus virus), compound cytotoxicity wells (cells plus compoundonly), compound colorimetric control wells (compound only), as well asexperimental wells (compound plus cells plus virus). Samples wereevaluated with triplicate measurements for antiviral efficacy andduplicate measurements for cytotoxicity. Six concentrations at half-logdilutions were used in order to determine the IC₅₀ values and to measurecellular cytotoxicity, if detectable.

At assay termination, the assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter Reagent, Promega) to determine cellviability and quantify compound cytotoxicity. MTS is metabolized by themitochondrial enzymes of metabolically active cells to yield a solubleformazan product, allowing the rapid quantitative analysis of cellviability and compound cytotoxicity. The MTS is a stable solution thatdoes not require preparation before use. At termination of the assay, 20μL of MTS reagent was added per well. The wells were incubated for fourto six hours at 37° C. Adhesive plate sealers were used in place of thelids, the seal plate was inverted several times to mix the solubleformazan product and the plate was read spectrophotometrically at490/650 nm with a Molecular Devices Vmax plate reader.

Using a computer program (Southern Research Institute, Frederick Md.),various values were determined including IC₅₀ (50%, inhibition of virusreplication), TC₅₀ (50% reduction in cell viability), and an antiviralindex (antiviral index=TC₅₀/IC₅₀). Values are provided in Table 1,below.

TABLE 1 Anti-HIV-1 Efficacy in CEM-SS Cells 26-m- 26-m- 15-m- di-15-PEG-3- PEG-7- PEG-7- m-PEG- O- O- NHCO- 7-NCO- Property SaquinavirSaquinavir Saquinavir Saquinavir Saquinavir In vitro 0.002 no activityno activity 0.05 0.56 Activity IC₅₀ (μM)Cytotoxicity >0.10 >1.00 >1.00 >1.00 >1.00 TC₅₀ (μM) Antiviral >26.3 —— >19.6 >1.8 Index

Example 4 Synthesis of PEG-Atazanavir

PEG-atazanavir was prepared. Schematically, the approach followed forthis example is shown below (compound numbers in bold in the schematiccorrespond to the compound numbers provided in the text of this Example4 alone).

Schematic for Synthesizing the Reagent

mPEG₃-SC-carbonate

Into a 100 mL flask was placed mPEG₃-OH (2.0 g, 12.1 mmol) and anhydrousdichloromethane (25 mL). The clear solution was cooled to 0° C., andthen triethylamine (1.86 mL, 13.4 mmol, 1.1 equivalents) was addedslowly. The solution was stirred for 15 minutes at 0° C., and then wasadded to a second flask containing a suspension of DSC (3.1 g, 12.1mmol) in dichloromethane (20 mL). The reaction mixture was allowed toequilibrate to room temperature. After approximately 18 hours, thelight-yellow reaction mixture was diluted with dichloromethane (60 mL),transferred to a separatory funnel, and partitioned with deionized water(100 mL). The aqueous layer was extracted with dichloromethane (4×80mL). The combined organics were washed with water, saturated sodiumbicarbonate, and saturated sodium chloride. The dried organic layer wasfiltered, concentrated under reduced pressure and dried overnight underhigh vacuum, to give 2.79 g (75%) of mPEG₃-SC-carbonate as a lightyellow oil. ¹H NMR (CDCl₃) δ 4.40 (m, 2H), 3.80 (m, 2H), 3.70 (bs, 6 H),3.60 (m, 2H), 3.35 (s, 3H), 2.80 (s, 4H); LC/MS=306 (M+1).

mPEG₅-SC-carbonate

Into a 100 mL flask was placed mPEG₅-OH (2.0 g, 7.92 mmol) and anhydrousdichloromethane (15 mL). The clear solution was cooled to 0° C., andthen triethylamine (1.32 mL, 9.51 mmol, 1.2 equivalents) was addedslowly. The solution was stirred for 15 minutes at 0° C., and then wasadded to a second flask containing a suspension of DSC (2.02 g, 7.92mmol) in dichloromethane (15 mL). The reaction mixture was allowed toequilibrate to room temperature. After approximately 18 hours, thelight-yellow reaction mixture was diluted with dichloromethane (40 mL),transferred to a separatory funnel, and partitioned with deionized water(80 mL). The aqueous layer was extracted with dichloromethane (4×50 mL).The combined organics were washed with water, saturated sodiumbicarbonate, and saturated sodium chloride. The dried organic layer wasfiltered, concentrated under reduced pressure and dried overnight underhigh vacuum, to give 2.59 g (83%) of mPEG₅-SC-carbonate as a lightyellow oil. ¹H NMR (CDCl₃) δ 4.45 (m, 2H), 3.75 (m, 2H), 3.68 (bs, 16H), 3.55 (m, 2H), 3.34 (s, 3H), 2.80 (s, 4H); LC/MS=394 (M+1).

mPEG₆-SC-carbonate

Into a 100 mL flask was placed mPEG₆-OH (2.0 g, 6.74 mmol) and anhydrousdichloromethane (12 mL). The clear solution was cooled to 0° C., andthen triethylamine (1.12 mL, 8.10 mmol, 1.2 equivalents) was addedslowly. The solution was stirred for 15 minutes at 0° C., and then wasadded to a second flask containing a suspension of DSC (1.73 g, 6.74mmol) in dichloromethane (15 mL). The reaction mixture was allowed toequilibrate to room temperature. After approximately 18 hours, thelight-yellow reaction mixture was diluted with dichloromethane (50 mL),transferred to a separatory funnel, and partitioned with deionized water(80 mL). The aqueous layer was extracted with dichloromethane (4×50 mL).The combined organics were washed with water, saturated sodiumbicarbonate, and saturated sodium chloride. The dried organic layer wasfiltered, concentrated under reduced pressure and dried overnight underhigh vacuum, to give 1.92 g (65%) of mPEG₆-SC-carbonate as a lightyellow oil. ¹H NMR (CDCl₃) δ 4.48 (m, 2H), 3.78 (m, 2H), 3.68 (bs, 20H), 3.58 (m, 2H), 3.38 (s, 3H), 2.84 (s, 4H); LC/MS=438 (M+1).

mPEG₇-SC-carbonate

Into a 100 mL flask was placed mPEG₇-OH (2.0 g, 5.87 mmol) and anhydrousdichloromethane (15 mL). The clear solution was cooled to 0° C., andthen triethylamine (1.22 mL, 8.81 mmol, 1.5 equivalents) was addedslowly. The solution was stirred for 15 minutes at 0° C., and then wasadded to a second flask containing a suspension of DSC (2.25 g, 8.81mmol) in dichloromethane (15 mL). The reaction mixture was allowed toequilibrate to room temperature. After approximately 18 hours, thelight-yellow reaction mixture was diluted with dichloromethane (50 mL),transferred to a separatory funnel, and partitioned with deionized water(80 mL). The aqueous layer was extracted with dichloromethane (4×50 mL).The combined organics were washed with water, saturated sodiumbicarbonate, and saturated sodium chloride. The dried organic layer wasfiltered, concentrated under reduced pressure and dried overnight underhigh vacuum, to give 2.82 g (90%) of mPEG₇-SC-carbonate as a lightyellow oil. ¹H NMR (CDCl₃) δ 4.45 (m, 2H), 3.78 (m, 2H), 3.65 (bs, 24H), 3.58 (m, 2H), 3.39 (s, 3H), 2.85 (s, 4H); LC/MS=482 (M+1).

mPEG₃-L-tert-Leucine

Into a 125 mL flask was placed L-tert-Leucine (0.43 g, 3.27 mmol) anddeionized water (12 mL). The solution was stirred for 30 minutes untilclear, followed by the addition of solid sodium bicarbonate (1.27 g,15.0 mmol, 4.6 equivalents). The cloudy solution was stirred at roomtemperature, under nitrogen. In a second flask the mPEG₃-SC-carbonate(1.24 g, 4.09 mmol, 1.25 equiv.) was taken up in deionized water (12 mL)and this solution was added all at once to the basic L-tert-Leucinesolution. The cloudy light-yellow reaction mixture was stirred at roomtemperature, under nitrogen. After approximately 20 hours, the clearmixture was cooled to 0° C., and carefully acidified with 2 N HCl to pH1 (20 mL). The acidic mixture was transferred to a separatory funnel andpartitioned with dichloromethane (50 mL) and additional water (50 mL).The aqueous layer was extracted with dichloromethane (4×50 mL). Thecombined organic layers were washed with water and saturated sodiumchloride, and dried over sodium sulfate. The dried organic layer wasfiltered, concentrated under reduced pressure and dried under highvacuum overnight, to give 0.83 g (79%) of mPEG₃-L-tert-Leucine as a paleyellow oil. ¹H NMR (CDCl₃) δ 5.45 (d, 1H), 4.26-4.35 (m, 2H), 4.14 (m,1H), 3.70 (bs, 17H), 3.65 (m, 2H), 3.32 (s, 3H), 0.96 (s, 9H); LC/MS=322(M+1).

mPEG₅-L-tert-Leucine

Into a 250 mL flask was placed L-tert-Leucine (0.68 g, 5.21 mmol) anddeionized water (20 mL). The solution was stirred for 30 minutes untilclear, followed by the addition of solid sodium bicarbonate (1.96 g,23.3 mmol, 4.5 equivalents). The cloudy solution was stirred at roomtemperature, under nitrogen. In a second flask the mPEG₅-SC-carbonate(3) was taken up in deionized water (20 mL) and this solution was addedall at once to the basic L-tert-Leucine solution. The cloudylight-yellow reaction mixture was stirred at room temperature, undernitrogen. After approximately 18 hours, the clear mixture was cooled to0° C., and carefully acidified with 2 N HCl to pH 1 (18 mL). The acidicmixture was transferred to a separatory funnel and partitioned withdichloromethane (50 mL) and additional water (50 mL). The aqueous layerwas extracted with dichloromethane (4×50 mL). The combined organiclayers were washed with water and saturated sodium chloride, and driedover sodium sulfate. The dried organic layer was filtered, concentratedunder reduced pressure and dried under high vacuum overnight, to give2.04 g (96%) of mPEG₅-L-tert-Leucine as a pale yellow oil. ¹H NMR(CDCl₃) δ 5.45 (d, 1H), 4.26-4.35 (m, 2H), 4.14 (m, 1H), 3.70 (bs, 17H),3.65 (m, 2H), 3.38 (s, 3H), 1.02 (s, 9H); LC/MS=410 (M+1).

mPEG₆-L-tert-Leucine

Into a 250 mL flask was placed L-tert-Leucine (0.45 g, 3.47 mmol) anddeionized water (15 mL). The solution was stirred for 30 minutes untilclear, followed by the addition of solid sodium bicarbonate (1.31 g,15.6 mmol, 4.5 equivalents). The cloudy solution was stirred at roomtemperature, under nitrogen. In a second flask the mPEG₆-SC-carbonate(1.9 gm, 4.34 mmol, 1.25 equiv.) was taken up in deionized water (15 mL)and this solution was added all at once to the basic L-tert-Leucinesolution. The cloudy light-yellow reaction mixture was stirred at roomtemperature, under nitrogen. After approximately 18 hours, the clearmixture was cooled to 0° C., and carefully acidified with 2 N HCl to pH1 (10 mL). The acidic mixture was transferred to a separatory funnel andpartitioned with dichloromethane (50 mL) and additional water (50 mL).The aqueous layer was extracted with dichloromethane (4×50 mL). Thecombined organic layers were washed with water and saturated sodiumchloride, and dried over sodium sulfate. The dried organic layer wasfiltered, concentrated under reduced pressure and dried under highvacuum overnight, to give 1.39 g (90%) of mPEG_(6-L)-tert-Leucine as apale yellow oil. ¹H NMR (CDCl₃) δ 5.47 (d, 1H), 4.10-4.30 (m, 2H), 4.14(m, 1H), 3.70 (bs, 20H), 3.65 (m, 2H), 3.38 (s, 3H), 1.02 (s, 9H);LC/MS=454 (M+1).

mPEG₇-L-tert-Leucine

Into a 250 mL flask was placed L-tert-Leucine (0.31 g, 2.32 mmol) anddeionized water (15 mL). The solution was stirred for 30 min untilclear, followed by the addition of solid sodium bicarbonate (0.89 g,10.6 mmol, 4.5 equivalents). The cloudy solution was stirred at roomtemperature, under nitrogen. In a second flask the mPEG₇-SC-carbonate(1.4 gm, 2.91 mmol, 1.25 equiv.) was taken up in deionized water (15 mL)and this solution was added all at once to the basic L-tert-Leucinesolution. The cloudy light-yellow reaction mixture was stirred at roomtemperature, under nitrogen. After approximately 18 hours, the clearmixture was cooled to 0° C., and carefully acidified with 2 N HCl to pH1 (8 mL). The acidic mixture was transferred to a separatory funnel andpartitioned with dichloromethane (50 mL) and additional water (50 mL).The aqueous layer was extracted with dichloromethane (4×50 mL). Thecombined organic layers were washed with water and saturated sodiumchloride, and dried over sodium sulfate. The dried organic layer wasfiltered, concentrated under reduced pressure and dried under highvacuum overnight, to give 1.0 g (85%) of mPEG₇-L-tert-Leucine as a paleyellow oil. ¹H NMR (CDCl₃) δ 5.46 (d, 1H), 4.10-4.25 (m, 2H), 4.14 (m,1H), 3.70 (bs, 24H), 3.65 (m, 2H), 3.38 (s, 3H), 1.02 (s, 9H); LC/MS=498(M+1).

Schematic for Synthesizing PEG-Atazanavir

Methods

All reactions with air- or moisture-sensitive reactants and solventswere carried out under nitrogen atmosphere. In general, reagents andsovents (except PEG-based reagents) were used as purchased withoutfurther purification. Analytical thin-layer chromagography was performedon silica F₂₅₄ glass plates (Biotage). Components were visulalized by UVlight of 254 nm or by spraying with phosphomolybdic acid. Flashchromatography was performed on Biotage SP4 system. ¹H NMR spectra:Bruker 300 MHz; chemical shifts of signals are expressed in parts permillion (ppm) and are referenced to the deuterated solvents used. MSspectra: rapid resolution Zorbax C18 column; 4.6×50 mm; 1.8 μm. HPLCmethod had the following parameters: column, Betasil C18, 5-μm (100×2.1mm); flow, 0.5 mL/minute; gradient, 0-23 minutes, 20% acetonitrile/0.1%TFA in water/0.1% TFA to 100% acetonitrile/0.1% TFA; detection, 230 nm.t_(R) refers to the retention time. Abbreviations: TPTU,O-(1,2-Dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate;DIPEA, N,N′-Diisopropylethylamine.

4-Pyridin-2-yl-benzaldehyde (3)

A mixture of 4-formyl-phenylboronic acid (5.0 g, 33.0 mmol) and2-bromopyridine (5.53 g, 35.0 mmol, 1.05 equiv.) in 265 mL of 4:3toluene/95% ethanol was degassed with nitrogen for 30 minutes and thenheated under a nitrogen atmosphere, resulting in a clear solution. Aslurry of Pd(PPh₃)₄ (0.77 g) in 50 mL of a 4:4 mixture of toluene and95% ethanol was added, followed by 50 mL of 3M aqueous Na₂CO₃. Theresulting mixture was gently refluxed at 77° C. After 16 hours, thereaction mixture was cooled to room temperature, and the solid removedby filtration. The filtrate was transferred to a separatory funnel, andthe layers separated. The aqueous layer was extracted with toluene (3×50mL). The combined organics were washed with water, then saturated sodiumchloride, and dried over sodium sulfate. The solution was filtered, andthe filtrate concentrated under reduced pressure to give a yellow oil.Purification by Biotage chromatography (40+M cartridge; gradient, 0 to5% methanol/dichloromethane) gave 4.13 g (68%) of (3) as a light-yellowsolid. TLC R_(f) (hexane/ethyl acetate, 2:1)=0.25; ¹H NMR (CDCl₃) δ 10.1(s, HCO), 8.77 (d, 1H), 8.20 (d, 2H), 8.00 (d, 2H), 7.81 (m, 2H), 7.31(q, 1H); MS (M)⁺=184; HPLC t_(R) 1.2 minutes.

N-1-(tert-Butyloxycarbonyl)-N-2-[4-(pyridine-2-yl)benzylidene]-hydrazone(4)

To a 100 mL flask was added (3) (0.50 g, 2.73 mmol), tert-butylcarbazate (0.36 g, 2.73 mmol), 2-propanol (3.0 mL) and toluene (3.0 mL).The mixture was heated to reflux (85° C.) under inert atmosphere for twohours, cooled to room temperature gradually and stirred overnight undernitrogen. After 16 hours the reaction mixture was filtered, and thefilter cake was washed with a cold mixture of toluene and hexane (1:3;100 mL). The cake was dried under vacuum to afford 0.73 g (90%) of (4)as an off-white solid. TLC R_(f) (hexane/ethyl acetate, 1:2)=0.38; ¹HNMR (CDCl₃) δ 8.70 (d, 1H), 8.02 (m, 3H), 7.87 (s, 1H), 7.81 (s, 1H),7.76 (m, 3H), 7.25 (m, 1H), 1.55 (s, 9H); MS (M)⁺=298; HPLC t_(R) 2.1minutes.

N′-(4-Pyridin-2-yl-benzyl)-hydrazinecarboxylic acid tert-butyl ester (5)

Into a 100 mL flask was placed (4) (0.45 g, 1.50 mmol) in THF (3.0 mL).To this solution was added 99% sodium cyanoborohydride (0.12 g, 1.80mmol, 1.2 equivalents), followed by a solution of p-TsOH (0.35 g, 1.80mmol, 1.2 equivalents) in THF (3.0 mL). After 1.5 hours, additionalp-TsOH (0.35 g, 1.80 mmol, 1.2 equivalents) in THF (3.0 mL) was added.After 16 hours at room temperature, the THF was removed under reducedpressure. The white residue was partitioned between ethyl acetate (35mL) and water (35 mL). The aqueous layer was extracted with ethylacetate (3×35 mL). The combined organics were washed with water, thensaturated sodium chloride, and then dried over sodium sulfate. Afterfiltration, concentration under reduced pressure, and drying under highvacuum for 6 h, 0.41 g (91%) of (5) was obtained as a white solid. TLCR_(f) (hexane/ethyl acetate, 1:2)=0.30; ¹H NMR (DMSO-d₆) δ 8.64 (d, 1H),8.26 (sb, HN), 8.02 (d, 2H), 7.93 (d, 1H), 7.85 (dd, 1H), 7.42 (d, 2H),7.32 (dd, 1H), 4.80 (m, HN), 3.92 (d, 2H), 1.38 (s, 9H); MS (M)⁺=300;HPLC t_(R) 7.0 minutes.

N′-(3-tert-Butoxycarbonylamino-2-hydroxy-4-phenyl-butyl)-N′-(4-pyridin-2-yl-benzyl)-hydrazinecarboxylicacid tert-butyl ester (7)

Into a 100 mL flask was placed (5) (1.0 g, 3.34 mmol), (6)(2S,3S)-1,2-epoxy-3-(Boc-amino)-4-phenylbutane (2.78 g, 10.5 mmol, 3.16equivalents), and 2-propanol (15 mL). The reaction was heated to reflux.After approximately 61 hours of refluxing, the heat was removed, and themixture cooled to room temperature. To the cooled mixture was addedwater/ice (50 mL). To the aqueous mixture was added dichloromethane (50mL) and then transferred to a separatory funnel. The aqueous layer wasextracted with dichloromethane (3×50 mL). The combined organics werewashed with water, then saturated sodium chloride, and then dried oversodium sulfate. The dried organic solution was filtered, and thefiltrate was concentrated under reduced pressure, and then dried underhigh vacuum overnight. The yellow foam was purified by Biotagechromatography (40+M cartridge; 0 to 5% methanol/dichloromethane over 25CV) to give 1.24 g (66%) of (7) as a white solid. TLC R_(f)(hexane/ethyl acetate, 1:2)=0.45; NMR (CD₃OD) δ 8.60 (d, 1H), 7.88 (m4H), 7.50 (d, 2H), 7.36 (m, 1H), 7.25 (m, 4H), 7.18 (m, 1H), 3.93 (m,2H), 3.70 (m, 2H), 3.0-2.6 (m, 4H), 1.33 (s, 9H), 1.30 (s, 9H); MS(M)⁺=563; HPLC t_(R) 9.6 minutes.

3-Amino-4-phenyl-1-[N-(4-pyridin-2-yl-benzyl)-hydrazino]-butan-2-oltrihydrochloride (8)

The Boc-aza-isostere (7) (1.2 g, 2.1 mmol) was taken up in 1,4-dioxane(16 mL), and stirred at room temperature, under nitrogen. After fiveminutes, 4N HCl (12 mL) was added via syringe. There was immediateprecipitate formation, and the mixture was stirred at room temperature,under nitrogen. After approximately 18 hours, the dioxane was removedunder reduced pressure. The yellow residue was azeotroped with toluene(3×25 mL), and then dried under high vacuum. After 6 hours under highvacuum, 0.92 g (91%) of (8) was obtained as a yellow solid. ¹H NMR(CD₃OD) δ 8.87 (d, 1H), 8.69 (m, 1H), 8.42 (d, 1H), 8.06 (m, 3H), 7.80(d, 2H), 7.28 (m, 6H), 4.25 (m, 3H), 3.13 (m, 2H), 2.88 (d, 2H); MS(M)⁺=472.

Synthesis of di-mPEG_(a)-Atazanavir

Synthesis of di-mPEG₃-Atazanavir

Into a 100 mL flask was placed mPEG₃-tert-Leucine (0.34 gm, 1.05 mmol,3.0 equivalents) in anhydrous dichloromethane (3 mL) and cooled to 0° C.Next, TPTU (0.31 gm, 1.05 mmol, 3.0 equiv.), and Hunigs base (0.36 mL,2.11 mmol, 6.0 equiv.) were added. The cloudy solution was stirred at 0°C. for 15 minutes, and then the diamino backbone trihydrochloride (8)(0.16 gm, 0.35 mmol) was added, as a solid, followed by adichloromethane rinse (3 mL). The ice bath was removed and the reactionmixture allowed to equilibrate to room temperature. After approximately20 hours, the reaction mixture was diluted with dichloromethane (20 mL).The mixture was transferred to a separatory funnel, and partitioned withdeionized water (50 mL). The aqueous layer was extracted withdichloromethane (4×30 mL). The combined organics were washed with water,saturated sodium bicarbonate, and saturated sodium chloride. The organiclayer was dried over sodium sulfate. The drying agent was filtered off,and the filtrate concentrated under reduced pressure to give a yellowoil. Purification was performed using Biotage (40+M cartridge; gradientelution: 0 to 5% methanol/dichloromethane) to give 0.14 gm (45%) ofdi-mPEG₃-Atazanavir as a clear oil. TLC R_(f) (5%methanol/dichloromethane)=0.22; ¹H NMR (CDCl₃) δ 8.71 (d, 1H), 7.98 (d,2H), 7.81 (m, 2H), 7.45 (d, 2H), 7.10-7.30 (m, 10H), 6.22 (d, 1H), 5.35(d, 1H), 4.25 (m, 4H), 4.01 (m, 4H), 3.50-3.80 (m, 24H), 3.38 (s, 3H),2.70-3.0 (m, 4H), 0.85 (d, 18H); MS (M)⁺=969; HPLC t_(R) 7.85 minutes.(96% purity).

di-mPEG₅-Atazanavir

Into a 100 mL flask was placed m-PEG₅-tert-Leucine (2.0 gm, 4.88 mmol,4.6 equiv.) in anhydrous dichloromethane (10 mL) and cooled to 0° C.Then added TPTU (1.45 gm, 4.88 mmol, 4.6 equiv.), and Hunigs base (1.85mL, 10.6 mmol, 10.0 equiv.) The cloudy solution was stirred at 0° C. for15 minutes, and then the diamino backbone trihydrochloride (8) (0.50 gm,1.06 mmol) was added, as a solid, followed by a dichloromethane rinse(10 mL). The ice bath was removed and the reaction mixture allowed toequilibrate to room temperature. After approximately 20 hours, thereaction mixture was diluted with dichloromethane (40 mL). The mixturewas transferred to a separatory funnel, and partitioned with deionizedwater (60 mL). The aqueous layer was extracted with dichloromethane(4×50 mL). The combined organics were washed with water, saturatedsodium bicarbonate, and saturated sodium chloride. The organic layer wasdried over sodium sulfate. The drying agent was filtered off, and thefiltrate concentrated under reduced pressure to give a yellow oil.Purification was performed using Biotage (40+M cartridge; gradientelution: 0 to 5% methanol/dichloromethane) to give 0.70 gm (58%) ofdi-mPEG₅-Atazanavir as a light-yellow oil. TLC R_(f) (5%methanol/dichloromethane)=0.23; ¹H NMR (CDCl₃) δ 8.60 (d, 1H), 7.88 (d,2H), 7.65 (m, 2H), 7.38 (d, 2H), 7.10-7.25 (m, 8H), 6.18 (d, 1H), 5.30(m, 2H), 4.15 (m, 4H), 3.92 (m, 3H), 3.45-3.65 (m, 40H), 3.30 (s, 3H),2.65-2.90 (m, 4H), 0.80 (d, 18H); MS (M)⁺=1146; HPLC t_(R) 7.72 minutes.(98% purity).

di-mPEG₆-Atazanavir

Into a 100 mL flask was placed mPEG₆-tert-Leucine (0.81 gm, 1.78 mmol,3.0 equiv.) in anhydrous dichloromethane (3 mL) and cooled to 0° C.Next, EDC (0.34 gm, 1.78 mmol, 3.0 equiv.) and HOBT (0.24 gm, 1.78 mmol,3.0 equiv.) were added. The cloudy solution was stirred at 0° C. for 15minutes, and then the diamino backbone trihydrochloride (8) was added(0.28 gm, 0.59 mmol), as a solid, followed by a dichloromethane rinse (5mL). The ice bath was removed and the reaction mixture allowed toequilibrate to room temperature. After approximately 28 hours, thereaction mixture was diluted with dichloromethane (35 mL). The mixturewas transferred to a separatory funnel, and partitioned with deionizedwater (60 mL). The aqueous layer was extracted with dichloromethane(4×50 mL). The combined organics were washed with water, saturatedsodium bicarbonate, and saturated sodium chloride. The organic layer wasdried over sodium sulfate. The drying agent was filtered off, and thefiltrate concentrated under reduced pressure to give a yellow oil.Purification was performed using Biotage (40+M cartridge; gradientelution: 0 to 5% methanol/dichloromethane) to give 0.27 gm (40%) ofdi-mPEG₆-Atazanavir as a clear oil. TLC R_(f) (5%methanol/dichloromethane)=0.17; ¹H NMR (CDCl₃) δ 8.75 (d, 1H), 78.02 (d,2H), 7.85 (m, 2H), 7.50 (d, 2H), 7.10-7.25 (m, 6H), 6.22 (d, 1H), 5.40(m, 2H), 4.20 (m, 4H), 4.15 (m, 3H), 3.52-3.70 (m, 48H), 3.38 (s, 3H),2.75-2.92 (m, 4H), 0.85 (d, 18H); MS (M)⁺=1234; HPLC t_(R) 7.70 min.(96% purity).

di-mPEG₇-Atazanavir: Into a 100 mL flask was placed mPEG₇-tert-Leucine(2.13 gm, 4.29 mmol, 4.6 equiv.) in anhydrous dichloromethane (10 mL)and cooled to 0° C. Then added TPTU (1.28 gm, 4.29 mmol, 4.6 equiv.),and Hunigs base (1.14 mL, 6.53 mmol, 7.0 equiv.) The cloudy solution wasstirred at 0° C. for 15 minutes, and then the diamino backbonetrihydrochloride (0.44 gm, 0.93 mmol) was added, as a solid, followed bya dichloromethane rinse (10 mL). The ice bath was removed and thereaction mixture allowed to equilibrate to room temperature. Afterapproximately 22 hours, the reaction mixture was diluted withdichloromethane (30 mL). The mixture was transferred to a separatoryfunnel, and partitioned with deionized water (50 mL). The aqueous layerwas extracted with dichloromethane (4×50 mL). The combined organics werewashed with water, saturated sodium bicarbonate, and saturated sodiumchloride. The organic layer was dried over sodium sulfate. The dryingagent was filtered off, and the filtrate concentrated under reducedpressure to give a yellow oil. Purification was performed using Biotage(40+M cartridge; gradient elution: 0 to 5% methanol/dichloromethane) togive 0.47 gm (38%) of di-mPEG₇-Atazanavir as a light-yellow oil. NMR(CDCl₃) δ 8.60 (d, 1H), 7.90 (d, 2H), 7.70 (m, 2H), 7.35 (d, 2H),7.10-7.25 (m, 8H), 6.12 (d, 1H), 5.30 (m, 2H), 4.10 (m, 4H), 3.92 (m,3H), 3.50-3.70 (m, 56H), 3.28 (s, 3H), 2.62-2.90 (m, 4H), 0.78 (d, 18H);MS (M)⁺=1321; HPLC t_(R) 7.69 min. (96% purity).

Example 5 Synthesis of PEG-Darunavir “Approach A”

PEG-darunavir was prepared using a first approach. Schematically, theapproach followed for this example is shown below (compound numbers inbold in the schematic correspond to the compound numbers provided in thetext of this Example 5 alone).

Synthesis of L-gulono-1,4-lactone (2)

A solution of L-ascorbic acid (23.1 g, 0.13 mol) in 170 ml of water washydrogenated using 10% Pd/C (2.2 g) in a Parr hydrogenator at 50° C. and50 psi hydrogen pressure for 24 hours. The catalyst was removed byfiltration and the water removed in vacuo to afford 23.2 g (0.13 mol,99%) of a white crystalline solid. Upon recrystallization frommethanol-ethyl acetate, 22.0 g of the desired product was obtained. ¹HNMR (DMSO): δ 5.80 (d, 1H), 5.30 (d, 1H), 4.95 (d, 1H), 4.65 (t, 1H),4.45 (m, 1H), 4.23-4.15 (m, 2H), 3.75 (m, 1H), 3.48 (m, 2H).

Synthesis of 5,6-isopropylidene-L-gulono-1,4-lactone (3)

A solution of L-gulono-1,4-lactone (11.08 g, 62.0 mmol) indimethylformamide (100 ml) was cooled to 10° C. and p-toluenesulfonicacid (0.09 g, 0.50 mmol) was added portionwise with stirring. To theresultant solution, isopropenyl methyl ether (5.83 g, 80.5 mmol) wasadded dropwise at 10° C. The cooling bath was removed and the solutionwas further stirred at room temperature for 24 hours. The solution wasthen treated with sodium carbonate decahydrate (11 g) and the suspensionwas vigorously stirred for two hours. The solid was removed byfiltration and mother liquor (filtrate) was concentrated using a rotaryevaporator. The yellow residue was recrystallized from toluene (25 ml).The product was isolated by suction, washed with hexane/ethanol (9:1, 50ml), and dried: yield of colorless crystalline (3): 11.22 g (82.7%). ¹HNMR (DMSO): δ 5.87 (d, 1H), 5.42 (br. 1H), 4.43 (t, 1H), 4.41-4.21 (m,3H), 4.06 (m, 1H), 3.75 (m, 1H), 1.35 (s, 3H), 1.30, (s, 3H).

Synthesis of E-R-3-(2,2-dimethyl-[1,3]-dioxolan-4-yl)-acrylic acidethylester (5)

To a well-stirred slurry of KIO₄ (10.60 g, 0.046 mol, 2.3 eq), KHCO₃(4.60 g, 0.046 mol, 2.3 equiv) in water (24 g) was added dropwise asolution of L-5,6-O-isopropylidene-gulono-1,4-lactone (4.37 g, 0.020mol) in water (2.70 g) and THF (22.90 g) during three hours at 32-34° C.The reaction mixture was stirred for 4.5 hours at 32° C. The reactionmixture was cooled to 5° C. and kept at this temperature for 14 hours.The solids were removed by filtration and the cake was washed with THF(3.0 mL) and with another portion of THF (4.0 mL) by reslurrying. To thefiltrate was added dropwise under stirring triethyl phosphonoacetate(TEPA, 3.90 g, 0.017 mol) during 25 minutes at 13-17° C. Subsequently,K₂CO₃ (16.80 g) was added portionwise during 30 minutes at 17-25° C. Thereaction mixture was stirred for another 17 hours at 20° C. The aqueousand THF phases were separated and the aqueous phase extracted twice with100 mL of toluene. The combined THF and toluene phases were concentratedin vacuo giving 2.80 g of a light yellow liquid. ¹H NMR indicated thepresence of E-R-3-(2,2-dimethyl-[1,3]dioxolan-4-yl)-acrylic acidethylester (5, 78%). Thus, the crude yield of (5) was 70% yield based on(3). Of the above residue, 0.50 g was purified by flash chromatographyon silica gel using 3/7 (v/v) ethyl acetate/n-heptane as the eluent.This gave 0.37 g of (5) with a purity of 96%. ¹H NMR (300 MHz, CDCl₃) δ6.79 (1H, dd, J=16.0, 5.3 Hz), 6.01 (1H, dd, J=16.0, 0.9 Hz), 4.58 (1H,q, J=6.0 Hz), 4.16-4.06 (3H, m), 3.58 (1H, t, J=7.6 Hz), 1.35 (3H, s),1.31 (3H, s), 1.20 (3H, t, J=7.0 Hz). ¹³C NMR (75 MHz, CDCl₃) δ 166.0(C), 144.7 (CH), 122.4 (CH), 110.2 (C), 75.0 (CH), 68.8 (CH₂), 60.6(CH₂), 26.5 (CH₃), 25.8 (CH₃), 14.2 (CH₃). LC-MS, calculated forC₁₀H₁₇O₄ (M+H⁺) 201.1. found 201.1.

(3aS,4S,6aR)-4-Methoxy-tetrahydro-furo[3,4-b]furan-2(3H)-one (α-7)

To 1.75 g of non-chromatographed (5) (78 wt % pure, 1.37 g, 6.80 mmol)was added to nitromethane (458 mg, 7.50 mmol) in 5.0 mL of methanol andthe solution was cooled to 10° C. Subsequently, DBU (1.03 g, 6.80 mmol)was added dropwise during 35 minutes at 10-21° C. After stirring for 18hours at 20° C. the resulting dark-red solution was cooled to 0° C. andNaOMe (15 mL of 0.50 M solution in methanol, 7.50 mmol) was addeddropwise over 30 minutes at 0° C. After 30 minutes stirring at 0° C. thereaction mixture was quenched into a solution of H₂SO₄ (2.43 g, 96%,23.80 mmol) in methanol (2.43 g) at 0-5° C. by dropwise addition duringthree hours under vigorous stirring. After two hours stirring at 0-2° C.the reaction mixture was quenched into a stirred slurry of KHCO₃ (3.53g) in water (6.80 mL) at 0-6° C. by dropwise addition during one hour.The pH was adjusted to 4.1 with H₂SO₄ (96%) at 0° C. After heating up to20° C. the salts were removed by filtration and washed with ethylacetate (3×3.75 mL). The wash liquor was used later on in theextractions. The mother liquor of the filtration was concentrated invacuo to remove the methanol. To the resulting residue was added water(0.80 g) and the pH was adjusted to 4.1 with H₂SO₄ (96%). The resultingaqueous solution was extracted with ethyl acetate (7.0 mL, 4×5.0 mL).The combined organic phases were concentrated in vacuo at 35-40° C. Thevolatiles were coevaporated with isopropanol (3×1.40 g) giving a residue(1.46 g) consisting of a crude mixture of (α-7) and (β-7), which wasdissolved in isopropanol (2.02 g) at 70° C. The insoluble material wasremoved and the filtrate was cooled resulting in spontaneouscrystallization of (α-7). The crystals were isolated by filtration,washed with isopropanol (2×1.0 mL, 0° C.) and dried in vacuo at 40° C.until a constant weight was achieved giving (α-7) as an off-whitecrystalline product [390 mg, 37% yield based on (5)]. The puritywas >99%. The mother liquor and wash liquors of the first (α-7)crystallization were concentrated in vacuo, methanol (1.20 mL) was addedand the resulting mixture concentrated in vacuo. Methanol (1.20 mL) wasadded once more and the mixture concentrated in vacuo again. To theresidue was added methanol (0.45 g) and methanesulfonic acid (MeSO₃H,0.027 g, 0.28 mmol) and the solution was heated to reflux. After onehour at reflux (60-65° C.), the solution was cooled to 33° C.,neutralized with triethylamine (0.029 g, 1.05 equiv based on MeSO₃H) andconcentrated in vacuo. To the resulting residue was added isopropanol(1.20 mL) and the mixture was concentrated in vacuo to give 0.88 g ofcrude product. The residue was dissolved in isopropanol (0.37 g) at 47°C. The resulting solution was cooled down to 2° C. during 2.5 hours. Thecrystalline product was isolated by filtration, washed with isopropanol(3×0.20 mL, 0° C.) and dried in vacuo at 40° C. until a constant weightwas achieved giving a second crop of (α-7) as an off-white crystallineproduct (0.098 g). The purity was >99%. Thus, the total yield of thefirst and second crop of (α-7) based on (5) was 46%.

The GC assay for compounds (α-7) and (β-7) was performed with an Agilent6890 GC (EPC) and a CP-Sil 5 CB column (part number CP7680 (Varian) orequivalent) of 25 mm and with a film thickness of 5 gm using a columnhead pressure of 5.1 kPa, a split flow of 40 mL/minute and an injectiontemperature of 250° C. The used ramp was: initial temperature 50° C. (5minutes), rate 10° C./minute, final temperature 250° C. (15 minutes).Detection was performed with an FID detector at a temperature of 250° C.The retention times were as follows: chlorobenzene (internal standard)17.0 minutes, (α-7) 24.9 minutes, (β-7) 25.5 minutes. The retention timeof (β-7) was determined by epimerizing pure (α-7) (as prepared above) toan approximately 3:1 mixture of (α-7) and (β-7) in methanol using 0.2equiv MeSO₃H at ambient temperature during 16 hours (¹H NMR and GC-MSconfirmed that only (β-7) had been formed). For the quantification of(β-7) it was assumed that the response factor of (β-7) was identical tothat of (α-7). ¹H NMR (300 MHz, CDCl₃) δ 5.15 (1H, dd, J=7.4, 3.8 Hz),4.88 (1H, s), 4.10 (1H, d, J=11.1 Hz), 3.96 (1H, dd, J=10.9, 3.8 Hz),3.33 (3H, s), 3.10-2.99 (1H, m), 2.84 (1H, dd, J=18.2, 11.0 Hz), 2.51(1H, dd, J=18.3, 3.7 Hz). ¹³C NMR (75 MHz, CDCl₃) M75.9 (C), 110.0 (CH),83.0 (CH), 70.6 (CH₂), 54.5 (CH₃), 45.1 (CH), 31.7 (CH₂). LC-MS:calculated for C₇H₁₁O₄ (M+H⁺) 159.06. found 159.06. e.e.>99% (asdetermined by GC).

(3R,3aS,6aR)-Hexahydro-furo[2,3-b]furan-3-ol (8)

To a solution of (α-7) (1.42 g, 9.0 mmol) in THF (8.0 g) was addeddropwise during 30 minutes a 10% solution of LiBH₄ (2.16 g, 1.1 equiv)and the reaction mixture was stirred at 50° C. for 2.5 hours. Theobtained suspension was cooled to −10° C. and a 32% aqueous HCl solution(1.36 g, 0.012 mol, 1.3 equiv based on LiBH₄) was added dropwise over aperiod of four hours keeping the temperature <−5° C. After stirring foran additional two hours at −10° C., triethylamine (1.325 g, 0.013 mol,1.1 equiv based on HCl) was added dropwise over one hour keeping thetemperature <0° C. The reaction mixture was warmed up and concentratedat atmospheric pressure to a residual weight of approximately 5.0 g, theresidue taken up in ethyl acetate (18.0 g) and concentrated once more atatmospheric pressure to a residual weight of approximately 5.0 g. Theresidue was taken up in ethyl acetate (18.0 g), stirred at reflux for 15minutes and cooled to 0° C. The salts were removed by filtration andwashed with cold (0° C.) ethyl acetate (2×1.5 g). The combined filtrateswere concentrated in vacuo at <40° C. to a colorless oil containing 0.94g of (8) [7.23 mmol, 80% based on (α-7), purity 87 wt % based on ¹HNMR]. The oil was purified by flash chromatography on silica gel usingethyl acetate as the eluent (R_(f)=0.56). This gave 0.89 g (6.85 mmol)of (8) with a purity of >99% which corresponds to 76% yield based on(α-7). ¹H NMR (300 MHz, DMSO-d₆) δ 5.52 (1H, d, J=4.8 Hz), 5.14 (1H, d,J=4.5 Hz), 4.27-4.17 (1H, m), 3.84-3.74 (2H, m), 3.72-3.62 (1H, m), 3.33(1H, dd, J=22.6, 14.1 Hz), 2.77-2.66 (1H, m), 2.24-2.14 (1H, m),1.75-1.59 (1H, m). ¹³C NMR (75 MHz, DMSO-d₆) δ 108.8 (CH), 72.1 (CH₂),69.4 (CH), 68.8 (CH₂), 45.8 (CH), 24.6 (CH₂). NMR (300 MHz, CDCl₃) δ5.62 (1H, d, J=4.9 Hz), 4.36 (1H, q, J=7.2 Hz), 3.94-3.77 (3H, m), 3.52(1H, dd, J=8.9, 7.1 Hz), 3.20 (1H, s), 2.84-2.73 (1H, m), 2.30-2.20 (1H,m), 1.87-1.72 (1H, m). ¹³C NMR (75 MHz, CDCl₃) δ 109.3 (CH), 72.7 (CH₂),70.4 (CH), 69.7 (CH₂), 46.3 (CH), 24.7 (CH₂). GC-MS: calculated forC₆H₁₁O₃ (M+H⁺) 131.0. found 131.0. e.e.>99% (as determined by GC). Thee.e. determination of 8 was performed with an HP 5890 GC and a Supelco24305 Betadex column of 60 mm and an internal diameter of 0.25 mm andwith a film thickness of 0.25 gm using a column head pressure of 30 psi,a column flow of 1.4 mL/minute, a split flow of 37.5 mL/minute and aninjection temperature of 250° C. The used ramp was: initial temperature80° C. (1 minute), rate 4° C./minute, final temperature 180° C. (5minutes). Detection was performed with an FID detector at a temperatureof 250° C. The retention times were as follows: (8) 27.1 min,(3S,3aR,6aS)-hexahydro-furo[2,3-b]furan-3-ol [the enantiomer of (8)]27.3 minutes. Racemic (8) required for the e.e. determination wasprepared according to the same procedure as described above foroptically active (8) except that racemic (α-7) was used as the startingmaterial.

Synthesis of Compound (9)

A solution of compound (8) (500 mg, 3.85 mmol) and N,N-disuccinimidyl(1.47 g, 5.75 mmol) in 20 mL of CH₃CN was added triethyl amine (1.10 mL,10.40 mmol). The resulting solution was stirred at room temperature for7 hours. The reaction mixture was concentrated under reduced pressure.The resulting residue was treated with 20 mL of saturated KHCO₃ and thenextracted with ethyl acetate (150 mL×3). The organic phase was washedwith water (150 mL×3) and dried over Na₂SO₄. Compound (9) (827 mg, yield79%) was obtained after removing the solvent and dried under vacuum. ¹HNMR (300 MHz, CDCl₃) δ 2.00 (m, 1H), 2.15 (m, 1H), 2.87 (br., 4H), 3.14(m, 1H), 3.96 (m, 2H), 4.03 (m, 1H), 4.12 (m, 1H) 5.28 (m, 1H), 5.76 (d,1H).

Synthesis of Compound (11)

To a stirred solution of compound (10) (962 mg, 3.65 mmol) in 2-propanol(40 mL) at room temperature was added isobutyl amine (1.60 g, 21.92mmol). The resulting mixture was reacted at 75° C. for six hours. Afterthis period, the reaction mixture was concentrated under reducedpressure. The resulting residue was dissolved in 5 ml of 2-propanol andconcentrated again under reduced pressure. The desired product wasobtained (1.17 g, yield: 95%) as a white solid; ¹H NMR (300 MHz, CDCl₃)δ 0.91 (d, 3H), 0.93 (d, 3H), 1.37 (s, 9H), 1.72 (m, 1H), 2.42 (d, 2H),2.70 (d, 2H), 2.86 (m, 1H), 3.01 (dd, 1H), 3.48 (m, 1H), 3.84 (br., 1H),4.74 (d, 1H), 7.20-7.33 (m, 5H); LC-MS (m/z) calcd. 336.25. found 337.25[M+H]+.

Synthesis of Compound (12)

To a stirred solution of the amine prepared above (1.16 g, 3.48 mmol) ina mixture of CH₂Cl₂ (30 mL) and saturated aqueous sodium bicarbonate (20mL) at 23° C. was added 4-nitrobenzenesulfonyl chloride (1.16 g, 5.21mmol). The resulting mixture was stirred at room temperature for 16hours. The mixture was then extracted with CH₂Cl₂ and dried overanhydrous Na₂SO₄. Removal of solvent under reduced pressure, followed bycolumn chromatography over silica gel (3% EtOAc in CH₂Cl₂ as theeluent), yielded compound (12) (1.29 g, 72%) as a white amorphous solid:¹H NMR (300 MHz, CDCl₃) 0.86 (d, 3H), 0.87 (d, 3H), 1.36 (s, 9H),1.84-1.92 (m, 1H), 2.86-2.95 (m, 2H), 2.98 (d, 2H), 3.19 (d, 2H),3.75-3.82 (m, 2H), 4.64 (d, 1H), 7.22-7.32 (m, 5H), 7.95 (d, 2H), 8.32(d, 2H); LC-MS (m/z) calcd., 521.22. found, 544.3 [M+Na]⁺.

Synthesis of Compound (13)

To a solution of compound (12) (1.28 g, 2.40 mmol) in EtOAc (20 mL) wasadded Pd/C (100 mg). The mixture was stirred at room temperature underan H₂ (15 psi) for 10 h. The reaction mixture was filtered over Celite,and the filter cake was washed with EtOAc. Removal of solvent underreduced pressure, followed by column chromatography on silica gel (7%EtOAc in CHCl₃ as the eluent) afforded the corresponding aromatic amine(1.16 g, 98%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 0.86 (d, 3H),0.89 (d, 3H), 1.34 (s, 9H), 1.86 (m, 1H), 2.77 (dd, 1H), 2.89-3.15 (m,5H), 3.85 (br., 2H), 4.05 (br., 1H), 4.17 (s, 2H), 4.65 (br., 1H), 6.71(d, 2H), 7.19-7.30 (m, 5H), 7.58 (d, 2H); LC-MS (m/z) calcd., 491.3.found: 492.3 [M+H]⁺, 514.23. [M+Na]⁺.

Synthesis of Compound (16) (General Procedure for 16a, 16b and 16c)

A solution of compound (13) (98 mg, 0.20 mmol) and mPEG_(n)-CHO (n=3, 5or 7, run separately) (0.30 mmol) in CH₃OH (10 mL) was stirred at 85° C.under azeotropic conditions for 90 minutes (4.0 ml of CH₃OH wasremoved). After this period, the reaction mixture was cooled to roomtemperature and sodium borohydride (20 eq.) was added in portions. Themixture was stirred at 50° C. for two hours, and then the reaction wasquenched with sodium bicarbonate. 150 ml of DCM was added. The solutionwas washed with H₂O (3×150 ml). The organic phase was dried over sodiumsulfate and was then concentrated under reduced pressure. The residuewas purified by column chromatography over silica gel (2% MeOH in CHCl₃as the eluent) to provide compound (16a), (16b) or (16c) respectively(yield, 70-80%) as colorless oil. Compound (16a) (n=3), ¹H NMR (300 MHz,CDCl₃) δ 0.86 (d, 3H), 0.89 (d, 3H), 1.33 (s, 9H), 1.82 (m, 1H), 2.77(dd, 1H), 2.89-2.92 (m, 2H), 2.99-3.11 (m, 3H), 3.75-3.80 (m, 2H), 3.32(m, 2H), 3.38 (s, 3H), 3.57 (m, 2H), 3.60-3.90 (m, 11H), 4.04 (br., 1H),4.62 (d, 1H), 4.85 (t, 1H), 6.60 (d, 2H), 7.19-7.30 (m, 5H), 7.54 (d,2H); LC-MS (m/z) calcd. 637.3. found: 638.3 [M+H]⁺. Compound (16b)(n=5), NMR (300 MHz, CDCl₃) δ 0.86 (d, 3H), 0.89 (d, 3H), 1.34 (s, 9H),1.80-1.86 (m, 1H), 2.77 (dd, 1H), 2.89-2.92 (m, 3H), 2.99-3.11 (m, 2H),3.32 (m, 2H), 3.36 (s, 3H), 3.54 (m, 2H), 3.58-3.90 (m, 19H), 4.65 (d,1H), 4.98 (t, 1H), 6.59 (d, 2H), 7.19-7.30 (m, 5H), 7.54 (d, 2H).Compound (16c) (n=7), ¹H NMR (300 MHz, CDCl₃) δ 0.86 (d, 3H), 0.89 (d,3H), 1.34 (s, 9H), 1.80-1.86 (m, 1H), 2.77 (dd, 1H), 2.89-3.11 (m, 5H),3.32 (m, 2H), 3.36 (s, 3H), 3.54 (m, 2H), 3.58-3.90 (m, 27H), 4.65 (d,1H), 4.98 (t, 1H), 6.59 (d, 2H), 7.19-7.30 (m, 5H), 7.54 (d, 2H).

Synthesis of Compound (17) (General Procedure for 17a, 17b and 17c)

A solution of compound (17a), (17b) or (17c) (each run separately)(0.151 mmol) in a mixture of 30% trifluoroacetic acid in CH₂Cl₂ (4 mL)was stirred for 60 min. After this period, the reaction mixture wasconcentrated under reduced pressure and the resulting residue wasredissolved in CH₂Cl₂ (5.0 mL). To this solution were added compound 9(45 mg, 0.17 mmol) and triethylamine (0.155 mL, 1.51 mmol). Theresulting mixture was stirred for 2 h. The reaction mixture was thenconcentrated under reduced pressure, and the residue was purified bycolumn chromatography over silica gel (2% MeOH in CHCl₃ as the eluent)to provide compound (17a), (17b), and (17c), respectively (yield:80-89%) as an oil. Compound (17a) (n=3), ¹H NMR (300 MHz, CDCl₃) δ 0.87(d, 3H), 0.93 (d, 3H), 1.42-1.46 (m, 1H), 1.57-1.65 (m, 1H), 1.79-1.85(m, 1H), 2.75-2.81 (m, 2H), 2.87-2.98 (m, 3H), 3.05-3.16 (m, 2H), 3.34(m, 2H), 3.38 (s, 3H), 3.58 (m, 2H), 3.64-3.74 (m, 10H), 3.82-4.00 (m,5H), 4.97-5.01 (m, 2H), 5.63 (d, 1H), 6.67 (d, 2H), 7.18-7.28 (m, 5H),7.53 (d, 2H); LC-MS (m/z) calcd: 693.3. found 694.3 [M+H]⁺. Compound(17b) (n=5), NMR (300 MHz, CDCl₃) δ 0.87 (d, 3H), 0.93 (d, 3H), 1.46 (m,1H), 1.60 (m, 1H), 1.82 (m, 1H), 2.75-2.81 (m, 2H), 2.87-2.98 (m, 3H),3.05-3.16 (m, 2H), 3.32 (m, 2H), 3.36 (s, 3H), 3.54 (m, 2H), 3.64-3.74(m, 18H), 3.82-3.92 (m, 5H), 4.97-5.01 (m, 2H), 5.63 (d, 1H), 6.67 (d,2H), 7.18-7.28 (m, 5H), 7.54 (d, 2H); LC-MS (m/z) calcd. 781.4. found782.5 [M+H]⁺. Compound (17c) (n=7), ¹H NMR (300 MHz, CDCl₃) δ 0.87 (d,3H), 0.92 (d, 3H), 1.46 (m, 1H), 1.60 (m, 1H), 1.82 (m, 1H), 2.75-2.81(m, 2H), 2.87-2.98 (m, 3H), 3.05-3.16 (m, 2H), 3.30 (m, 2H), 3.36 (s,3H), 3.54 (m, 2H), 3.64-3.74 (m, 26H), 3.82-3.92 (m, 5H), 4.97-5.05 (m,3H), 5.63 (d, 1H), 6.62 (d, 2H), 7.18-7.28 (m, 5H), 7.53 (d, 2H); LC-MS(m/z) calcd: 869.4. found 870.3 [M+H]⁺.

Example 6 Synthesis of PEG-Darunavir “Approach B”

PEG-darunavir was prepared using a second approach. Schematically, theapproach followed for this example is shown below (compound numbers inbold in the schematic correspond to the compound numbers provided in thetext of this Example 6 alone).

Synthesis of Compound (18)

To a stirred solution of compound (10) (264 mg, 1.0 mmol) [prepared inaccordance with the procedure for synthesizing compound (10) in Example12] in 2-propanol (10 mL) at 23° C. was added mPEG₃-NH₂ (489 mg, 3.0mmol). The resulting mixture was stirred at 75° C. for six hours. Afterthis period, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (biotage, CH₃OH/DCM, 4-15% CH₃OH, 20 CV). 390 mg of correspondingamine (18) was obtained (yield, 91.5%) as sticky oil. ¹H NMR (300 MHz,CDCl₃) δ 1.35 (s, 9H), 1.85-1.89 (m, 1H), 2.70 (m, 1H), 2.86 (m, 4H),3.00 (dd, 1H), 3.35 (s, 3H), 3.54-3.75 (m, 10H), 3.85 (m, 1H), 4.70 (d,1H), 7.10-7.40 (m, 5H); LC-MS (m/z) calcd., 426.3. found 427.2 [M+H]⁺.

Synthesis of Compound (19)

To a stirred solution of above prepared amine (18) (390 mg, 0.92 mmol)in a mixture of CH₂Cl₂ (15 mL) and saturated aqueous sodium bicarbonate(10 mL) at 23° C. was added 4-nitrobenzenesulfonyl chloride (304 mg,1.38 mmol). The resulting mixture was stirred at room temperature for 16hours. The mixture was then extracted with CH₂Cl₂ and dried overanhydrous Na₂SO₄. Removal of solvent under reduced pressure, followed bycolumn chromatography over silica gel (biotage, DCM/CH₃OH, CH₃OH: 1-6%,20 CV) gave the desired product (19) (455 mg, 81%) as sticky oil: NMR(300 MHz, CDCl₃) δ 1.37 (s, 9H), 2.85 (m, 1H), 3.10 (m, 2H), 3.30 (m,1H), 3.41 (m, 2H), 3.38 (s, 3H), 3.50-3.85 (m, 11H), 3.90 (m, 1H), 4.45(d, 1H), 4.95 (d, 1H), 7.22-7.32 (m, 5H), 7.95 (d, 2H), 8.32 (d, 2H).LC-MS (m/z) calcd., 611.3. found, 612.3 [M+H]⁺.

Synthesis of Compound (20)

To a solution of compound (19) (455 mg, 0.74 mmol) in EtOAc (10 mL) wasadded Pd/C (40 mg, 10%). The mixture was stirred at room temperatureunder an H₂ atmosphere (30 psi) for 4.0 hours. The reaction mixture wasfiltered over Celite, and the filter cake was washed with EtOAc. Removalof solvent under reduced pressure afforded the corresponding aromaticamine (420 mg, 98%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 1.36 (s,9H), 2.90-3.10 (m, 3H), 3.10-3.30 (m, 3H), 3.37 (s, 3H), 3.56 (m, 2H),3.63-3.90 (m, 11H), 4.54 (br., 1H), 4.88 (d, 1H), 6.65 (d, 2H),7.19-7.30 (m, 5H), 7.53 (d, 2H); LC-MS (m/z), calcd., 581.3. found:582.3 [M+H]⁺.

Synthesis of Compound (21)

A solution of compound (20) (116 mg, 0.2 mmol) in a mixture of 30%trifluoroacetic acid in CH₂Cl₂ (4 mL) was stirred at room temperaturefor 1.0 hour. After this period, the reaction mixture was concentratedunder reduced pressure and the residue was redissolved in CH₂Cl₂ (5.0mL). To this solution were added(3R,3aS,6aR)-3hydroxyhexahydrofuro[2,3-b]furanyl succinimidyl carbonate[compound (9)] (54 mg, 0.2 mmol) and triethylamine (0.5 mL). Theresulting mixture was stirred for two hours. At which point, thesolution was concentrated under reduced pressure. The resulting residuewas purified by column chromatography (biotage, DCM/CH₃OH, CH₃OH: 2-6%,20 CV) to provide compound (21) (102 mg, 80%) as a oil. ¹H NMR (300 MHz,CDCl₃) δ 1.40-1.60 (m, 1H), 1.60-1.80 (m, 1H), 1.90 (br., 1H), 2.75 (m,1H), 2.90 (m, 1H), 3.00-3.15 (m, 2H), 3.15-3.30 (m, 3H), 3.37 (s, 3H),3.50-3.85 (m, 12H), 3.85-3.98 (m, 4H), 4.23 (br., 2H), 4.50 (br., 1H),5.02 (m, 1H), 5.40 (d, 1H), 5.64 (d, 1H), 6.67 (d, 2H, J) 8.6 Hz),7.18-7.28 (m, 5H), 7.51 (d, 2H); LC-MS (m/z), calcd., 637.2. found,638.2 [M+H]⁺.

Example 7 Synthesis of PEG-Darunavir “Approach C”

PEG-darunavir was prepared using a third approach. Schematically, theapproach followed for this example is shown below (compound numbers inbold in the schematic correspond to the compound numbers provided in thetext of this Example 7 alone).

Synthesis of Compound (23)

To a stirred solution of Boc-Tyr-OMe [compound (22), 10.33 g, 0.035 mol]and potassium carbonate (7.20 g, 0.052 mol) in acetone (45 mL) was addedBnBr (6.00 g, 0.035 mol). The resulting mixture was stirred at 60° C.for 16 hours. After this period, the solid was removed by filtration andthe reaction mixture was concentrated under reduced pressure. Theresulting residue was purified by column chromatography (biotage:DCM/CH₃OH, CH₃OH, 0-6%, 15 CV). Product (23) was obtained (13.0 g, 96%)as a white solid; ¹H NMR (300 MHz, CDCl₃) δ 1.44 (s, 9H), 3.05 (m, 2H),3.72 (s, 3H), 4.55 (m, 1H), 5.00 (m, 1H), 5.05 (s, 2H), 6.90 (d, 2H),7.10 (d, 2H), 7.20-7.38 (m, 5H); LC-MS (m/z) calcd., 385.2. found 408.2[M+Na]⁺.

Synthesis of Compound (25)

Compound (23) (12.74 g, 0.033 mol) and iodochloromethane (23.35 g, 0.132mol) in anhydrous THF (150 ml) was cooled to −78° C. and LDA (83 ml,0.165 mol) was added dropwise. Upon completion of the addition, thesolution was stirred at −75° C. for an additional 15 minutes. An aceticacid solution (20 ml of THF+20 ml of HOAc) was added dropwise whilekeeping the temperature below −70° C. Stirring was continued for 15minutes after addition of 200 ml of toluene, then 100 ml of 1% HCl wasadded. The organic phase was washed with 0.5 M NaHCO₃ (10 ml) andseparated. The solution was added 100 ml of ethanol and cooled to −78°C. NaBH₄ (6.3 g, 0.17 mol) was added. The mixture was stirred at −78° C.for 1 hour and then the reaction was quenched by addition of 100 ml ofsaturated KHSO₄. The organic phase was washed with water and dried overNa₂SO₄. The resulting solid, after solvent removal, was washed withhexane and then recrystallized from ethyl acetate. Compound (25a) [3.5g, 30% based on compound (23)] was obtained as yellow solid. ¹H NMR (300MHz, CDCl₃) δ 1.39 (s, 9H), 2.91 (m, 2H), 3.17 (br., 1H), 3.57 (m, 1H),3.67 (m, 1H), 3.84 (m, 2H), 4.57 (m, 1H), 5.05 (s, 2H), 6.92 (d, 2H),7.13 (d, 2H), 7.20-7.38 (m, 5H); LC-MS (m/z) calcd., 405.2. found 428.2[M+Na]⁺.

Synthesis of Compound (26)

Compound (25a) (2.18 g, 5.38 mmol) was suspended in a 0.1 N solution ofpotassium hydroxide in methanol (5.92 mmol, 59.2 ml). The resultingmixture was stirred at 50° C. for 1.5 hours. The solvent was removedunder reduced pressure and the solid was dissolved in 100 ml DCM, whichwas subsequently washed with water (100 mL×3). The solution was driedand solvent was removed under reduced pressure. The desired product wasobtained as a yellow solid (1.74 g, 88%) was obtained. ¹H NMR (300 MHz,CDCl₃) δ 1.40 (s, 9H), 2.77 (m, 3H), 2.92 (m, 2H), 3.65 (br., 1H), 4.44(br., 1H), 5.05 (s, 2H), 6.92 (d, 2H), 7.15 (d, 2H), 7.26-7.38 (m, 5H);LC-MS (m/z) calcd., 369.2. found, 370.2 [M+H]+, 392.2 [M+Na]⁺.

Synthesis of Compounds (27) & (28)

To a stirred solution of compound (26) (1.74 g, 4.80 mmol) in 2-propanol(60 mL) at 23° C. was added isobutyl amine (2.20 g, 30 mmol). Theresulting mixture was reacted at 75° C. for 6 hours. After this period,the reaction mixture was concentrated under reduced pressure. Theresidue was dissolved in 5 ml of 2-propanol and concentrated again underreduced pressure. Compound (27) was obtained (1.97 g) as a yellow solid,which was used in next reaction without further purification.

To a stirred solution of compound (27) (1.97 g, 4.45 mmol) in a mixtureof CH₂Cl₂ (40 mL) and saturated aqueous sodium bicarbonate (30 mL) at23° C. was added 4-nitrobenzenesulfonyl chloride (1.48 g, 6.67 mmol).The resulting mixture was stirred at room temperature for 16 hours. Themixture was then extracted with CH₂Cl₂ (150 mL×2). The organic phase waswashed with water (150 mL×3) and dried over anhydrous Na₂SO₄. Removal ofsolvent under reduced pressure, followed by column chromatography(biotage: DCM/CH₃OH, CH₃OH, 1-6%, 15CV, 6-8% 5CV), yielded compound (28)(2.14 g, 77%) as a white amorphous solid: ¹H NMR (500 MHz, CDCl₃) δ 0.87(d, 3H), 0.89 (d, 3H), 1.37 (s, 9H), 1.87 (m, 1H), 2.86 (m, 2H), 2.99(d, 2H), 3.19 (d, 2H), 3.72 (m, 1H), 3.79 (m, 2H), 4.61 (d, 1H), 5.05(s, 2H), 6.90 (d, 2H), 7.14 (d, 2H), 7.35 (m, 1H), 7.44 (m, 4H), 7.95(d, 2H), 8.34 (d, 2H); LC-MS (m/z) calcd., 627.26. found, 650.3 [M+Na]⁺.

Synthesis of Compound (29)

To a solution of compound (28) (2.14 g, 3.41 mmol) in THF (20 mL) wasadded Pd/C (428 mg). The mixture was stirred at room temperature underan H₂ atmosphere (45 psi) for 48.0 hours. The reaction mixture wasfiltered over Celite, and the filter cake was washed with THF. Removalof the solvent under reduced pressure afforded the correspondingaromatic amine (1.48 g, 86%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ0.86 (d, 3H), 0.90 (d, 3H), 1.36 (s, 9H), 1.85 (m, 1H), 2.77 (m, 1H),2.84 (m, 1H), 2.90 (m, 2H), 2.92 (d, 1H), 3.07 (m, 1H), 3.71 (m, 1H),3.77 (m, 1H), 4.16 (br., 2H), 4.72 (d, 1H), 6.66 (d, 2H), 6.75 (d, 2H),7.09 (d, 2H), 7.52 (d, 2H).

General Procedure for the Synthesis of Compounds (30a), (30b) and (30c)

A solution of compound (29) (152 mg, 0.30 mmol) and mPEG_(n)-Br (n=3, 5and 7, in three separate runs) (0.45 mmol) in acetone (10 mL) wasstirred at 70° C. for 20 hours. After this period, the reaction mixturewas cooled to room temperature and 150 mL of DCM was added. The solutionwas washed with water (150 mL×2). The organic phase was dried oversodium sulfate and then concentrated under reduced pressure. Theresulting residue was purified by column chromatography (biotage:DCM/CH₃OH, CH₃OH, 3-6%, 15CV, 6-8% 5CV) to provide compound (30a), (30b)and (30c), respectively (yield, 70-80%) as colorless oil. Compound (30a)(n=3): ¹H NMR (500 MHz, CDCl₃) δ 0.83 (d, 3H), 0.89 (d, 3H), 1.36 (s,9H), 1.82 (m, 1H), 2.62 (m, 1H), 2.69 (m, 1H), 2.86 (m, 1H), 2.92 (m,3H), 3.36 (s, 3H), 3.53 (m, 2H), 3.62 (m, 2H), 3.68 (m, 2H), 3.74 (m,4H), 3.85 (m, 3H), 4.08 (m, 2H), 4.40 (br., 2H), 4.77 (d, 1H), 6.62 (d,2H), 6.82 (d, 2H), 7.14 (d, 2H), 7.38 (d, 2H); LC-MS (m/z) calcd.,653.3. found, 654.4 [M+H]⁺. Compound (30b) (n=5): ¹H NMR (500 MHz,CDCl₃) δ 0.83 (d, 3H), 0.89 (d, 3H), 1.37 (s, 9H), 1.81 (m, 1H), 2.60(m, 2H), 2.85 (m, 1H), 2.92 (m, 3H), 3.35 (s, 3H), 3.52 (m, 2H),3.61-3.65 (m, 11H), 3.70 (m, 2H), 3.75 (m, 5H), 3.85 (m, 2H), 4.07 (m,2H), 4.49 (br., 2H), 4.73 (d, 1H), 6.62 (d, 2H), 6.82 (d, 2H), 7.15 (d,2H), 7.34 (d, 2H); LC-MS (m/z) calcd., 741.4. found, 742.5 [M+H]⁺,764.4. [M+Na]⁺. Compound (30c) (n=7): ¹H NMR (500 MHz, CDCl₃) δ 0.82 (d,3H), 0.89 (d, 3H), 1.36 (s, 9H), 1.80 (m, 1H), 2.85 (m, 1H), 2.92 (m,3H), 3.35 (s, 3H), 3.52 (m, 2H), 3.61-3.65 (m, 19H), 3.70 (m, 2H), 3.75(m, 5H), 3.85 (m, 2H), 4.07 (m, 2H), 4.49 (s, 2H), 4.73 (d, 1H), 6.61(d, 2H), 6.82 (d, 2H), 7.14 (d, 2H), 7.35 (d, 2H); LC-MS (m/z) calcd.,829.4. found, 830.5 [M+H]+.

General Procedure for the Synthesis of Compounds (31a), (31b) and (31c)

A solution of compound (30a), (30b) and (30c) (0.20 mmol, in threeseparate runs) in a mixture of 30% trifluoroacetic acid in CH₂Cl₂ (4.0mL) was stirred at room temperature for 40 minutes. After this period,the reaction mixture was concentrated under reduced pressure and theresidue was redissolved in CH₂Cl₂ (5.0 mL). To this solution were added(3R,3aS,6aR)-3 hydroxyhexahydrofuro[2,3-b]furanyl succinimidyl carbonate(54 mg, 0.20 mmol) and triethylamine (0.155 mL, 1.51 mmol). Theresulting mixture was stirred for one hour. The reaction mixture wasthen concentrated under reduced pressure, and the residue was purifiedby column chromatography (biotage, DCM/CH₃OH, CH₃OH: 0-4%, 20 CV, 4-6%,10 CV) to provide compounds (31a), (31b), and (31c), respectively(yield: 75-80%) as colorless oil. Compound (31a) (n=3): ¹H NMR (500 MHz,CDCl₃) δ 0.83 (d, 3H), 0.88 (d, 3H), 1.58 (m, 1H), 1.64 (m, 1H), 1.77(m, 1H), 2.65 (m, 1H), 2.72 (m, 2H), 2.90 (m, 2H), 2.98 (m, 2H), 3.35(s, 3H), 3.52 (m, 2H), 3.60 (m, 2H), 3.64 (m, 3H), 3.69 (m, 4H), 3.81(m, 5H), 3.92 (m, 1H), 4.03 (m, 2H), 4.46 (s, 2H), 5.00 (m, 1H), 5.16(d, 1H), 5.62 (d, 1H), 6.60 (d, 2H), 6.78 (d, 2H), 7.08 (d, 2H), 7.38(d, 2H); LC-MS (m/z) calcd: 709.3. found 710.3 [M+H]⁺. Compound (31b)(n=5): ¹H NMR (500 MHz, CDCl₃) δ 0.86 (d, 3H), 0.92 (d, 3H), 1.71-1.85(m, 3H), 2.65 (m, 2H), 2.78 (m, 1H), 2.97 (m, 4H), 3.36 (s, 3H), 3.54(m, 2H), 3.64 (m, 10H), 3.68 (m, 3H), 3.75 (m, 4H), 3.69 (m, 4H), 3.85(m, 4H), 3.90 (m, 1H), 4.00 (m, 1H), 4.10 (m, 2H), 4.50 (br., 2H), 5.06(m, 1H), 5.12 (d, 1H), 5.66 (d, 1H), 6.64 (d, 2H), 6.82 (d, 2H), 7.13(d, 2H), 7.37 (d, 2H); LC-MS (m/z) calcd: 797.4. found 798.4 [M+H]⁺.Compound (31c) (n=7), ¹H NMR (500 MHz, CDCl₃) δ 0.86 (d, 3H), 0.92 (d,3H), 1.71-1.85 (m, 3H), 2.62 (m, 2H), 2.78 (m, 1H), 2.97 (m, 4H), 3.37(s, 3H), 3.54 (m, 2H), 3.64 (m, 19H), 3.68 (m, 3H), 3.73 (m, 4H), 3.85(m, 4H), 3.90 (m, 1H), 4.00 (m, 1H), 4.08 (m, 2H), 4.52 (br., 2H), 5.06(m, 1H), 5.12 (d, 1H), 5.66 (d, 1H), 6.64 (d, 2H), 6.82 (d, 2H), 7.13(d, 2H), 7.37 (d, 2H); LC-MS (m/z) calcd: 885.4. found 886.5 [M+H]⁺.

Example 8 Synthesis of PEG-Tipranavir

PEG-tipranavir was prepared. Schematically, the approach followed forthis example is shown below (wherein Xa stands for oxazolidinone andcompound numbers in bold in the schematic correspond to the compoundnumbers provided in the text of this Example 8 alone).

In carrying out this synthesis, the following materials were used.Calcium hydride (CaH₂), ethylene glycol, trimethyl orthoacetate, sodiumhydroxide, titanium (IV) chloride, N,N-diisopropylethylamine (DIPEA),perchlorid acid 60% (HCLO₄), phenethylmagnesium chloride (1.0 M in THF),butyaldehyde, pyridinium chlorochromate (PCC), titanium (IV)isopropoxide, potassium tert-butoxide (KOBut), palladium/carbon (10 wt%), oxalyl chloride [(COCl)₂], dimethylsulfoxide (DMSO), anhydrousmethanol, sodium bronohydride (NaBH₄), and pyridine were purchased fromSigma-Aldrich (St Louis, Mo.). mPEG_(n)-OH (n=3, 5, 7) were purchasedfrom TCI America. 5-Trifluoromethyl-2-pyridinesulfonyl chloride waspurchased from Toronto Research Chemicals, Inc. (North York, ON,Canada). DCM was distilled from CaH₂. Tetrahydrofuran (THF) and otherorganic solvents were used as they purchased. 2-(E)-pentenoic acid,thionyl chloride, (R)-(−)-4-phenyl-2-oxazolidinone, n-butyl lithium (1M,Hexane), 3-bis(trimethylsilyl)amino]phenylmagnesium chloride (1.0 M,THF), copper bromide (I)-dimethyl sulfide, benzyl bromide, and ammoniumchloride were purchased from Sigma-Aldrich (St Louis, Mo.). Ammoniumhydroxide, sodium sulfate, ethyl acetate, and hexane were purchased fromFisher Scientific (Fair Lawn, N.J.). Magnesium sulfate, sodiumbicarbonate, and sodium carbonate were purchased from EM Science(Gibbstown, N.J.). DCM was distilled from CaH₂. THF (anhydrous) andacetonitrile were also purchased from Sigma-Aldrich and used aspurchased.

Acid Chloride Preparation (2A)

In a 100-mL flask equipped with a reflux condenser, 2-(E)-pentenoic acid(15.4 mL, 152 mmol) was added under N₂. After the reaction flask was setup in a water bath, thionyl chloride (10.5 mL, 144 mmol) was then addedslowly and the reaction was kept in the water bath for an additional tenminutes before it was removed and allowed to warm to room temperature.The reaction was kept at room temperature overnight and then heated to110° C. (external) in oil bath for 30 minutes and was kept at thistemperature for an additional 30 minutes. The solution was cool downbelow 40° C. before vacuum distillation was started. Vacuum distillationprovided desired product 2 (13.8 g, 81% yield) as a colorless liquid,under 45-55° C. (external)/8 mmHg. ¹H NMR (300 MHz, CDCl₃) δ 1.13 (t,3H, J=7.5 Hz), 2.29-2.39 (m, 2H), 6.07 (dt, 1H, J=1.5, 15.3 Hz), 7.28(dt, 1H, J=6.3, 15.3 Hz).

Oxazolidinone Amide Bond Formation (4A)

Oxazolidinone (3A) (6.90 g, 42.3 mmol) was added to a 500-mL flaskprotected with N₂ and was filled with anhydrous THF (265 mL). The THFsolution was cooled down to −78° C. in a dry-ice bath. Then n-BuLi (1.6M in hexane, 27.8 mL, 44.4 mmol) was added slowly (about 12 minutes).The reaction was kept at this temperature for 30 minutes before2-(E)-pentenoic acid chloride (2A) (5.51 g, 46.5 mmol) was added slowlyover seven minutes. The dry-ice bath was immediately removed afteraddition of the acid chloride was completed and the reaction solutionwas warmed to room temperature over 40 minutes. The reaction then wasquenched by a saturated solution of NH₄Cl (400 mL). A small amount ofpure de-ionized water was added to dissolve the precipitation of NH₄Cl.The organic THF phase was separated and the aqueous phase was extractedwith EtOAc (100 mL×2). The organic phases were combined, dried overMgSO₄, and concentrated to about 25 mL. While stirring, hexane (200 mL)was added and the crude product precipitated in a few minutes. Afterfiltration, the solution was concentrated to about 10 mL andprecipitated a second time with hexane (about 180 mL). The mother liquorwas concentrated and the resulting residue was purified on Biotage(EtOAc/Hex 6-50% in 20 CV). Three portions of colorless product (4A)were combined (9.95 g, 96% yield). R_(f)=0.45 (Hex:EtOAc=3:1), RP-HPLC(betasil C18, 0.5 mL/min, 60-100% ACN in 8 min) 7.40 min, LC-MS (ESI,MH⁺) 246.1. ¹H NMR (300 MHz, CDCl₃) δ 1.08 (t, 3H, J=7.5 Hz), 2.28 (p,2H, J=6.3 Hz), 4.28 (dd, 1H, J=3.9, 9.0 Hz), 4.70 (t, 1H, J=8.7 Hz),5.49 (dd, 1H, J=3.9, 8.7 Hz), 7.09-7.18 (m, 1H), 7.23-7.42 (m, 6H).

Asymmetric Michael Addition:

In a 500-mL flask protected with N₂, copper bromide (I)-dimethyl sulfide(7.44 g, 36.2 mmol) was added followed by anhydrous THF (75 mL). Thesolution was cooled down to −45° C. with dry-ice/acetonitrile before3-[bis(trimethylsilyl)amino]-phenylmagnesium chloride (1.0 M, 36.2 mL,36.2 mmol) was added dropwise over 30 minutes. The reaction was kept ata temperature between −40° C. to 0° C. for 20 minutes. A solution ofabove starting material (4A) (7.1 g, 29.0 mmol) in THF (19.3 mL) wasadded dropwise over 20 minutes. The reaction then was warmed to 0° C.over 10 min and then further to room temperature over 15 minutes. Thereaction mixture was quenched with the addition of aqueous NH₄Cl (70 mL)at room temperature for 15 minutes. The aqueous phase was then adjustedto pH=8 by addition of NH₄OH (5 mL). The solution was then poured intoan ether solution (250 mL) and the aqueous phase was separated. Theether phase was washed with NaHCO₃ (80 mL×2) until the aqueous phase wasnot blue to pH paper anymore. The ether phase was then dried over Na₂SO₄and concentrated in vacuo. The resulting residue was loaded on thereverse phase column (40 M×3, about 8 g crude each) and purified via20-70% ACN in 20 CV. Fractions were collected and acetonitrile wasevaporated. The aqueous phase then extracted with DCM (50 mL×3). Theorganic solution was combined, dried over Na₂SO₄, concentrated to giveproduct (6A) (8.73 g, 89% yield). R_(f)=0.11 (Hex:EtOAc=3:1), RP-HPLC(betasil C18, 0.5 mL/min, 60-100% ACN in 8 min) 5.67 min, LC-MS (ESI,MH⁺) 339.2. ¹H NMR (500 MHz, CDCl₃) δ 0.76 (t, 3H, J=7.2 Hz), 1.50-1.68(m, 2H), 2.90-3.00 (m, 1H), 3.06 (dd, 1H, J=7.2, 15.6 Hz), 3.48 (dd, 1H,J=7.5, 15.6 Hz), 4.17 (dd, 1H, J=4.2, 9.3 Hz), 4.64 (t, 1H, J=9.0 Hz),5.38 (dd, 1H, J=3.9, 8.7 Hz), 6.51-6.61 (m, 3H), 6.99-7.07 (m, 3H),7.22-7.28 (m, 3H).

Benzyl Protection of Amine:

The above product (6A) (13.5 g, 40 mmol) was dissolved in DCM (146 mL)and H₂O (106 mL) in a 500-mL flask. Solid sodium carbonate (25 g, 240mmol) and benzyl bromide (19.0 mL, 160 mmol) were added. The solutionwas heated (52° C. external) to reflux overnight (20 hrs) before it waschecked by TLC. The reaction was diluted with NaHCO₃ (300 mL) and DCMwas separated from the solution. The aqueous phase was then extractedwith DCM (60 mL×2) and organic phases were combined. The solution wasdried over Na₂SO₄ and concentrated. The residue was loaded on Biotage(40 M×2, 14 g crude each) over 6-22% EtOAc/Hex in 18 CV. The productfractions were collected and evaporated to generate a colorless softsolid product (2) (17.5 g, 84%). R_(f)=0.42 (Hex:EtOAc=3:1), RP-HPLC(betasil C18, 0.5 mL/min, 60-100% ACN in 8 min, 100% 8-12 min) 9.80 min,LC-MS (ESI, MH⁺) 519.2. ¹H NMR (300 MHz, CDCl₃) δ 0.65 (t, 3H, J=7.2Hz), 1.40-1.55 (m, 2H), 2.84-2.94 (m, 1H), 3.02 (dd, 1H, J=7.2, 15.6Hz), 3.42 (dd, 1H, J=7.5, 15.6 Hz), 4.15 (dd, 1H, J=3.9, 8.7 Hz),4.53-4.67 (m, 5H), 5.35 (dd, 1H, J=3.9, 8.7 Hz), 6.50-6.61 (m, 3H),6.98-7.07 (m, 3H), 7.18-7.29 (m, 13H).

Synthesis of Glycol Ortho Ester, Compound (3)

A fresh CaH₂ distilled starting material (26.3 g, 219 mmol) was mixedwith ethylene glycol (11 mL, 197 mmol) at room temperature. H₂SO₄ (3-4drops, 0.25%) was added and stirring at this temperature. A water sprayvacuo system with a solid NaOH dry bottle and a mercury manometer wasset up to the distillation reaction system. The vacuo was adjusted below95 mmHg (not less than 55 mmHg) and the temperature was graduallyincreased (10° C. per ten minutes). After a forerun (˜2 g) wascollected, a colorless product (16.2 g, 70% yield) was collected under68-71° C./58-60 mmHg. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (3H, s), 3.28 (3H,s), 3.97-4.12 (4H, m).

TiCl₄ Activated C—C Conjugation to Prepare Compound (4)

A pre-vacuo dried starting material (2) (6.45 g, 12.4 mmol) wasdissolved in DCM (50 mL) under the protection of N₂. It was then allowedto cool down to −78° C. in a dry-ice/acetone bath. TiCl₄ (2.45 mL, 22.3mmol) was dropwise added and the reaction at this temperature was keptfor five minutes before DIPEA (4.11 mL, 23.6 mmol) was added. The bathwas moved away immediately and the reaction was warm up to 0° C. insalt-ice bath. The enolate formation was kept at this temperature for 30minutes before it was re-cooled down to −78° C. Glycol ortho ester (3)(3.66 mL, 31 mmol) was added slowly. After addition, the reaction waswarm up to 0° C. and kept at this temperature for 2.5 hours. Thereaction was quenched with half saturated NH₄Cl and water. The solutionwas diluted with water and extracted with DCM (50 mL×3). The combinedorganic phase was washed with NaHCO₃ and dried over Na₂SO₄. TLC show thereaction was clean but ˜10% starting material remaining. Biotagepurification (40 M X5 times) provided a colorless product (5.47 g, 73%yield) product without contamination. R_(f)=0.51 (Hex:EtOAc=3:1), LC-MS(ESI, MH⁺) 605.3. ¹H NMR (300 MHz, CDCl₃) δ_(—)δ 0.55 (3H, t, J=7.2 Hz),0.86 (3H, s), 1.40-1.51 (2H, m) 2.89 (1H, dt, J=3.6, 11.1 Hz), 3.03 (1H,q, J=6.9 Hz), 3.44-3.50 (1H, m), 3.54 (1H, q, J=6.9 Hz), 3.62-3.72 (1H,m), 4.26 (1H, dd, J=3.6, 9.0 Hz), 4.55-4.67 (5H, m), 4.80 (1H, d, J=10.8Hz), 5.46 (1H, dd, J=3.3, 8.4 Hz), 6.59-6.63 (3H, m), 7.08 (1H, t, J=7.5Hz), 7.19-7.37 (15H, m).

Acid Hydrolysis of Acetal to Form Compound (5)

The acetal product (4) (5.47 g, 9.06 mmol) was dissolved in anhydrousTHF (18 mL). Deionized water (3.6 mL) and HClO₄ (3.6 mL) were added. Thereaction was started in an oil bath at the temperature of 40° C.(external) for 2.5 hours. After cooling down to room temperature, thesolution was neutralized with NaHCO₃ slowly to pH=8-9. The mixturesolution was diluted with water (100 mL) and extracted with DCM (80mL×3). The organic phase was dried over Na₂SO₄ and concentrated invacuo. The residue was loaded on Biotage column (25M) with gradientelute (4-13% EtOAc/Hex in 16 CV). A colorless solid (5.18 g, >100%yield) was collected after high vacuo drying. R₁=0.43 (Hex:EtOAc=3:1),RP-HPLC (betasil C18, 0.5 mL/min, 60-100% ACN in 10 min) 6.40 min, LC-MS(ES¹, MH⁺) 561.3. ¹H NMR (300 MHz, CDCl₃) δ 0.61 (3H, t, J=7.2 Hz), 1.63(3H, s), 1.07 (1H, dt, J=3.3, 10.8 Hz), 4.22 (1H, dd, J=3.9, 8.7 Hz),4.61 (4H, s), 4.67 (1H, t, J=9.0 Hz), 4.98 (1H, d, J=10.5 Hz), 5.42 (1H,dd, J=3.6, 8.7 Hz), 6.54-6.64 (3H, m), 7.09 (1H, t, J=8.1 Hz), 7.21-7.39(15H, m).

Synthesis of Compound (6)

Phenyl ethyl magnesium chloride (1M in THF, 120 mmol) was cannulated toa 500-mL flask together with THF (180 mL). The above mixture solutionwas then cool down to 0° C. using an ice-water bath before butyraldehyde(10.2 mL, 114 mmol) was added dropwise. TLC indicated a clean reactionafter one hour at this temperature. The reaction was then quenched withNH₄Cl (150 mL) and the THF was separated. The THF solution was washedwith saturated brine before it was dried over Na₂SO₄ and concentrated invacuo. Over 20 grams of the secondary alcohol product (>100% yield) wasobtained without further purification.

The secondary alcohol product (4.56 g, 25.6 mmol) was mixed with DCM(128 mL) at room temperature. PCC (6.62 g, 30.7 mmol) was added. Thereaction was kept at room temperature for two hours. Due to the TLCindicated an about 15% of remaining starting material, another part ofPCC (1.11 g, 5.1 mmol) was added and the reaction was finished in twohours. The solution mixture was filtrated though a layer of celite andsilica gel. The filtrated solution was then evaporated and the residuewas purified on Biotage column (40S). A colorless compound (6) (2.79 g,yield 62%) was collected. NMR proton spectrum indicated a product withimpurity <1%. ¹H NMR (300 MHz, CDCl₃) δ 0.89 (3H, t, J=7.2 Hz),1.56-1.63 (2H, m), 2.37 (2H, t, J=7.2 Hz), 2.72 (2H, t, J=7.2 Hz), 2.90(2H, t, J=7.5 Hz), 7.17-7.21 (3H, m), 7.26-7.28 (2H, m).

Ti-Activated C—C Conjugation, Aldol Reaction to Form Compound (7)

In a N₂ protected 100-mL flask, freshly distilled DCM (22 mL) was added.Ti(OPr)₄ (373 μL, 1.27 mmol) and TiCl₄ (377 μL, 3.44 mmol) were added inthat order. The reaction solution was cooled down to −78° C. in anacetone-dry ice bath and compound (5) (1.93 g, 3.44 mmol) in DCM (6 mL)solution was added slowly. The solution was reddish and was kept at thistemperature for five minutes before DIPEA (899 μL, 5.16 mmol) was added.The acetone-dry ice bath was taken away and warmed to 0° C. before anice-water bath was used. The enolate formation was kept at 0° C. for onehour before it was re-cooled down to −78° C. in an acetone-dry ice bath.Compound (6) (1.21 mL, 6.88 mmol) was added slowly. The solution wasthen warm up to 0° C. and kept at this temperature via ice-water bathfor one hour. The reaction was quenched by saturated NH₄Cl solution (30mL) and a diluted mixture was extracted by DCM (40 mL×3). The combinedorganic phase was then dried over Na₂SO₄ and concentrated in vacuo. Theresidue was loaded on the Biotage column (40S) with a gradient (8-18%EtOAc/Hex in 16 CV). A yellowish product (1.90 g, yield 75%) wascollected. R_(f)=0.42 (Hex:EtOAc=3:1), RP-HPLC (betasil C18, 0.5mL/minute, 60-100% ACN in ten minutes) 9.13 minutes, LC-MS (ESI, MO737.5.

Basic Hydrolysis and Lactonization to Synthesize Compound (8)

The aldol product (7) mixture (1.68 g, 2.28 mmol) was dissolved in theTHF (50 mL) under a N₂ atmosphere. After the sample was dissolved, thesolution was allowed to cool in ice-water bath for five minutes beforeKOBu^(t) (1 M, 2.74 mL) was added. The reaction was kept at thistemperature for 20 minutes. It was quenched with NH₄Cl (50 mL) and theorganic phase was diluted with EtOAc (150 mL). The aqueous phase wasthen separated (ensuring a pH<7) and the ether phase was washed withsaturated brine (50 mL). It was then dried over Na₂SO₄ and concentratedunder vacuo. The dried residue was then loaded on Biotage column (25 M)and purified (6-22% EtOAc/Hex in 16 CV) four times. The yellowish benzylamine compound (8) (712.1 mg, yield 54.5%) was solidified after highvacuo drying. R_(f)=0.41 (Hex:EtOAc=3:1), RP-HPLC (betasil C18, 0.5mL/minute, 60-100% ACN in ten minutes) 5.23 minutes, LC-MS (ESI, MH⁺)574.4.

Pd/C hydrogenation to synthesize(R)-3-(R)-1-(3-aminophenyl)propyl)-5,6-dihydro-4-hydroxy-6-phenethyl-6-propylpyran-2-one(9)

The benzyl amine compound (8) (265.8 mg, 0.464 mmol) was dissolved inEtOAc (6.5 mL) and MeOH (6.5 mL) mixture solution. The solution vial wasbubbling N₂ for exchange at least 15 minutes before catalyst addition.Stirring was stopped and the Pd/C catalyst (43 mg, 8 wt %×2) was addedslowly (or in small portions). The system was evacuated and rechargedwith hydrogen gas (<50 psi) three times (stop stirring during vacuo).The hydrogenolysis was then kept at room temperature under 50 psi forovernight (16 hrs) to complete. After release the pressure, the reactionmixture was first checked with HPLC to see the completeness before afiltration was performed. The catalyst residue and filter paper werecarefully washed with methanol. The solution was then evaporated andvacuo drying to give oil-like compound (9) (182 mg, 100% yield). Nofurther purification is necessary. RP-HPLC (betasil C18, 0.5 mL/minute,10-100% ACN in 8 minutes) 4.58 minutes, LC-MS (ESI, MO 394.2.

Preparation of Compound (10) via Swern Oxidation of mPEGn-OH

In a 250-mL flask with N₂ protection, DCM (105 mL) and oxalyl chloride(2M, 7.5 mL, 15 mmol) was added. The solution was cool down to −78° C.in dry-ice acetone bath for five minutes before DMSO (1.42 mL, 20.0mmol) was added. It was stirred vigorously at this temperature for 20minutes before a mPEG₇-OH (3.40 g, 10.0 mmol) and DCM (10 mL) mixturewas added. The reaction was kept at this temperature for another 20minutes before TEA (5.5 mL, 39.6 mmol) were added. The reaction was keptin dry-ice bath for three minutes and the bath was removed to graduallywarm up to ambient temperature for 25 minutes. It was quenched withsaturated NaHCO₃ (70 mL) and DCM solution was diluted (120 mL). Theorganic phase was separated and aqueous phase was extracted with DCM (20mL×2). It was dried over Na₂SO₄ and then concentrated and a slightyellow liquid with some solid inside (2.78 g, 82% yield) was saved inN₂. NMR indicated a 64% conversion mixture. Biotage FCC (3-10% MeOH inDCM in 16 CV) provided pure product for reductive amination. R_(f)=0.32(DCM:MeOH=10:1), NMR (300 MHz, CDCl₃) δ 3.39 (s, 3H), 3.54-3.57 (m, 2H),3.66 (s, 20H), 3.72-3.75 (m, 2H), 4.17 (s, 2H), 9.74 (s, 1H).

mPEG₅-CHO was synthesized in a similar approach. Crude product showed86% aldehyde with 99% yield. Biotage FCC (3-10% MeOH in DCM in 16 CV)provided 75% aldehyde product with 56% yield and 15% aldehyde mixturewith 25% yield. R_(f)=0.34 (DCM:MeOH=10:1), NMR (300 MHz, CDCl₃) δ 3.38(s, 3H), 3.38-3.57 (m, 2H), 3.67 (s, 11H), 3.70-3.75 (m, 3H), 4.17 (s,2H), 9.74 (s, 1H).

Reductive Amination to Synthesize Compound (11)

Compound (9) (69.6 mg, 0.177 mmol) was dissolved in methanol (3.4 mL).While stirring, mPEG₅-CHO (235 mg, 75% purity, 0.708 mmol) was addeddropwise. The reaction was run for 18 minutes and thereafter moved to awater bath at ambient temperature. NaBH₄ (54 mg, 1.42 mmol) was added inseveral portions. HPLC was used to check the reaction after threeminutes and evidenced the reaction achieved 77% conversion. The reactionwas quenched with NaHCO₃ (10′ mL) and diluted with water and EtOAc. Theorganic phase was then separated and dried over Na₂SO₄. HPLC show thereaction has 81% conversion with 13% of starting material remaining. Thesolution was diluted with NaHCO₃ aqueous solution and extracted with DCM(30 mL×3). The combined organic solution was evaporated to provide crudesample (178 mg). It was dissolved in ACN (6 mL) and water (2 mL) andpurified on AKTA (40-57% in 5 CV×2, 12.10 minutes). The acetonitrilesolution of collected product was evaporated and saturated with NaCl. Itwas extracted with DCM (30 mL×3) and combined solution was dried overNaSO₄, filtrated, concentrated under vacuo. A slightly yellowish product(75.9 mg, 69% yield) was obtained with purity over 99%. RP-HPLC (betasilC18, 0.5 mL/minute, 30-100% ACN in ten minutes) 5.53 minutes, LC-MS(ESI, MH⁺) 628.2.

This synthetic procedure was followed except mPEG₃-CHO was substitutedfor mPEG₅-CHO. With excess aldehyde (1.6 eq), the product mixture showed72% of conversion after work up. AKTA purification (40-50% ACN in 3 CV,13.2 minutes) provided 42% yield product with purity over >99%. RP-HPLC(betasil C18, 0.5 mL/minutes, 30-100% ACN in ten minutes) 5.65 minutes,LC-MS (ESI, MH⁺) 540.3.

This synthetic procedure was followed except mPEG₇-CHO was substitutedfor mPEG₅-CHO. With excess aldehyde (4.5 eq), the product mixture showed78% of conversion after work up. AKTA purification (40-57% in 5 CV)provided 73% yield of pure product (>99%). RP-HPLC (betasil C18, 0.5mL/minute, 60-100% ACN in eight minutes) 5.06 minutes, LC-MS (ESI, MH⁺)716.4.

Synthesis of Compound (13a)

The above AKTA purified product (11a) (96.8 mg, 0.180 mmol) wasdissolved in DCM (1.6 mL). After dissolving, the solution was cool downin an ice-water bath and trifluoropyridine sulphonyl chloride (48.6 mg,0.198 mmol) was added. Pyridine (44 μL, 0.54 mmol) was then added andthe reaction was warm up during the overnight reaction. HPLC showed theretention time of starting material was completed and the reaction wasquenched with NH₄Cl (10 mL). It was diluted with DCM and the separatedorganic phase was washed with brine. The organic phase was then driedover Na₂SO₄ and concentrated. The crude product (159.4 mg) was purifiedon Biotage (10-50% EtOAc in Hex with 16 CV) provided a slightlyyellowish product (13a) (73.1 mg) and a less pure product (35.7 mg) withthe total yield about 62%. R_(f)=0.22 (Hex:EtOAc=1:1), RP-HPLC (betasilC18, 0.5 mL/minute, 60-100% ACN in 8 minutes) 4.20 minutes, LC-MS (ESI,MH⁺) 749.3.

Following a procedure similar to the synthesis of compound (13a),compound (11b) (154.9 mg) produced the desired product (13b) (36.0 mg,93% pure) and a mixture of product (71.2 mg) with a yield of −52%.Purification over Biotage silical gel column (1-7% MeOH in DCM in 16CV). R_(f)=0.54 (EtOAc), RP-HPLC (betasil C18, 0.5 mL/minute, 60-100%ACN in 8 minutes) 4.36 minutes, LC-MS (ESI, MH⁺) 837.4.

Following a procedure similar to the synthesis of compound (13a),compound (11c) (167.7 mg) produced the desired product (13c) (39.9 mg,95% pure) and a mixture of product (82.3 mg) with a yield of ˜50%.Purification over Biotage silical gel column (2-7% MeOH in DCM in 16CV). R_(f)=0.25 (EtOAc), RP-HPLC (betasil C18, 0.5 mL/minute, 60-100%ACN in 8 minutes) 3.78 minutes, LC-MS (ESI, MH⁺) 925.5.

Example 9 Evaluation of Conjugates

Following the standard procedures for evaluating activity and efficacyin cell and cell-free assays (and as set forth in, for example, Example3), atazanavir conjugates (see Example 4), tipranivir conjugates (seeExample 8) and darunavir conjugates (see Examples 5 through 7). Theresults are shown in the tables 2 through 8 below.

TABLE 2 Anti-HIV-1 Efficacy in CEM-SS Cells CEM-SS/HIV-1_(RF) CEM-SSTherapeutic Compound EC₅₀ (μM) TC₅₀ (μM) Index AZT 0.001 >1.0 >1000.0Atazanavir 0.012 54.9 4578.2 di-mPEG-3- 0.038 95.7 2519.3 Atazanavirdi-mPEG-5- 1.1 >200.0 >181.0 Atazanavir di-mPEG-6- 2.5 158.6 62.4Atazanavir di-mPEG-7- 8.4 >200.0 >24.0 Atazanavir

TABLE 3 CELL-FREE ASSAY IC₅₀ IC₉₀ IC₅₀- IC₉₀ fold Compound (nM) (nM)fold change change Atazanavir 6.92 31.77 1.0 1.0 di-mPEG-3- 12.72 53.671.8 1.7 Atazanavir di-mPEG-5- 17.33 47.62 2.5 1.5 Atazanavir di-mPEG-6-15.63 67.82 2.3 2.1 Atazanavir di-mPEG-7- 10.92 39.93 1.6 1.3 Atazanavir

TABLE 4 CEM-SS/HIV-1RF EC₅₀- EC₅₀ TC₅₀ Therapeutic fold Compound (μM)(μM) Index change 0.001 >1.0 >1000.0 Atazanavir 0.012 54.9 4578.2 1di-mPEG-3- 0.038 95.7 2519.3 3.2 Atazanavir di-mPEG-5- 1.1 >200.0 >181.091.7 Atazanavir di-mPEG-6- 2.5 158.6 62.4 208.3 Atazanavir di-mPEG-7-8.4 >200.0 >24.0 700 Atazanavir

TABLE 5 Tipranavir Conjugates in Cell-Based Assay CEM-SS/HIV-1_(RF)CEM-SS Therapeutic Compound EC₅₀ (μM) TC₅₀ (μM) Index AZT0.002 >1.0 >500.0 Tipranavir 0.12 30.6 263.8 mPEG₃-amide-Tipranavir 0.081.3 15.5 mPEG₅-amide-Tipranavir >200.0 12.8 —mPEG₇-amide-Tipranavir >200.0 14.5 —

TABLE 6 Tipranavir Conjugates in Cell-Free Assay Compound IC₅₀ (nM)Saquinavir 0.079 Tipranavir 7.2 mPEG₃-amide-Tipranavir —mPEG₅-amide-Tipranavir — mPEG₇-amide-Tipranavir 12.2

TABLE 7 Darunavir Conjugates in Cell-Based Assay CEM-SS/ HIV-1_(RF)CEM-SS Therapeutic Compound EC₅₀ (μM) TC₅₀ (μM) Index AZT0.005 >1.0 >200.0 Darunavir <0.0007 118.8 >180,000.0 mPEG₃-Darunavir<0.0007 93.9 >142,272.7 mPEG₅-Darunavir 0.011 40.7 3697.3mPEG₇-Darunavir 0.014 167.6 11,974.9

TABLE 6 Darunavir Conjugates in Cell-Free Assay Compound IC₅₀ (nM)Darunavir 1.5 mPEG₃-Darunavir 5.3 mPEG₅-Darunavir 1.1 mPEG₇-Darunavir5.2 Saquinavir 0.001

1. A compound comprising a residue of a small molecule proteaseinhibitor covalently attached to a water-soluble, non-peptidic oligomer.2. The compound of claim 1, wherein the small molecule proteaseinhibitor is from the azahexane derivative class of small moleculeprotease inhibitors.
 3. The compound of claim 1, wherein the smallmolecule protease inhibitor is from the amino acid derivative class ofsmall molecule protease inhibitors.
 4. The compound of claim 1, whereinthe small molecule protease inhibitor is from the non-peptidicderivative class of small molecule protease inhibitors.
 5. The compoundof claim 1, wherein the small molecule protease inhibitor is from thepyranone class of small molecule protease inhibitors.
 6. The compound ofclaim 1, wherein the small molecule protease inhibitor is from thepentan-1-amine derivative class of small molecule protease inhibitors.7. The compound of claim 1, wherein the small molecule proteaseinhibitor is from the hexan-2-ylcarbamate derivative class of smallmolecule protease inhibitors.
 8. The compound of claim 1, wherein thesmall molecule protease inhibitor is from the sulfonamide derivativeclass of small molecule protease inhibitors.
 9. The compound of claim 1,wherein the small molecule protease inhibitor is from thetri-substituted phenyl derivative class of small molecule proteaseinhibitors.
 10. The compound of claim 1, wherein the small moleculeprotease inhibitor is selected from the group consisting of amprenavir,atazanavir, fosamprenavir, indinavir, lopinavir, saquinavir, nelfinavir,ritonavir, tipranovir, and darunavir.
 11. The compound of claim 1,wherein the small molecule protease inhibitor is selected from the groupconsisting of DMP-323, DMP-450, BMS186613, SC-55389a, and BILA 1096 BS12. The compound of claim 1, wherein the water-soluble, non-peptidicoligomer is a poly(alkylene oxide).
 13. The compound of claim 12,wherein the poly(alkylene oxide) is a poly(ethylene oxide).
 14. Thecompound of claim 1, wherein the water-soluble, non-peptidic oligomer ismade of between 1 and 30 monomers.
 15. The compound of claim 14, whereinthe water-soluble, non-peptidic oligomer is made of between 1 and 10monomers.
 16. The compound of claim 12, wherein the poly(alkylene oxide)includes an alkoxy or hydroxy end-capping moiety.
 17. The compound ofclaim 1, wherein a single water-soluble, non-peptidic oligomer iscovalently attached to the residue of the small molecule proteaseinhibitor.
 18. The compound of claim 1, wherein more than onewater-soluble, non-peptidic oligomer is covalently attached to theresidue of the small molecule protease inhibitor.
 19. The compound ofclaim 1, wherein the residue of small molecule protease inhibitor iscovalently attached via a stable linkage.
 20. The compound of claim 1,wherein the residue of the small molecule protease inhibitor iscovalently attached via a degradable linkage.
 21. The compound of claim1, wherein the residue of the small molecule protease inhibitor iscovalently attached to the water-soluble, non-peptidic oligomer via anether linkage.
 22. The compound of claim 1, wherein the residue of thesmall molecule protease inhibitor is covalently attached to thewater-soluble, non-peptidic oligomer via an amide linkage
 23. Thecompound of claim 1, wherein the residue of the small molecule proteaseinhibitor is covalently attached to the water-soluble, non-peptidicoligomer via a carbamate linkage.
 24. The compound of claim 1, whereinthe residue of the small molecule protease inhibitor is covalentlyattached to the water-soluble, non-peptidic oligomer via an aminelinkage.
 25. A composition comprising a compound comprising a residue ofa small molecule protease inhibitor covalently attached to awater-soluble, non-peptidic oligomer, and optionally, a pharmaceuticallyacceptable excipient.
 26. A composition of matter comprising a compoundcomprising a residue of a small molecule protease inhibitor covalentlyattached to a water-soluble, non-peptidic oligomer, wherein the compoundis present in a dosage form.
 27. A method comprising covalentlyattaching a water-soluble, non-peptidic oligomer to a small moleculeprotease inhibitor.
 28. A method comprising administering a compoundcomprising a residue of a small molecule protease inhibitor covalentlyattached to a water-soluble, non-peptidic oligomer.